Methods for treatment of atherosclerosis

ABSTRACT

Disclosed herein are methods and compositions for preventing or treating atherosclerosis in a mammalian subject. The methods comprise administering to the subject an effective amount of an aromatic-cationic peptide and in some applications, a second active agent chemically linked to the peptide, to subjects in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ApplicationNo. 61/678,992 filed on Aug. 2, 2012, U.S. Application No. 61/695,807filed on Aug. 31, 2012, and U.S. Application No. 61/695,850 filed onAug. 31, 2012. The content of each application is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present technology relates generally to compositions and methods ofpreventing or treating atherosclerosis. In particular, embodiments ofthe present technology relate to administering aromatic-cationicpeptides in effective amounts to prevent or treat atherosclerosis inmammalian subjects.

SUMMARY

The present technology relates to the treatment or prevention ofatherosclerosis in mammals through the administration of atherapeutically effective amount of aromatic-cationic peptides and, insome embodiments, a second active agent. In some embodiments, the secondactive agent includes an antihyperlipidemic drug. In some embodiments,the second active agent includes a statin. In some embodiments, thesecond active agent and the aromatic-cationic peptide are chemicallylinked.

In one aspect, the present disclosure provides a pharmaceuticalcomposition comprising (i) a peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or apharmaceutically acceptable salt thereof, such as acetate ortrifluoroacetate salt, and (ii) a second active agent, e.g., anantihyperlipidemic agent. In some embodiments, the second active agentcomprises a statin. In some embodiments, the peptide and the secondactive agent are chemically linked.

In one aspect, the present disclosure provides a method for treatingatherosclerosis in a mammalian subject, the method comprisingadministering an effective amount of peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂or a pharmaceutically acceptable salt, such as acetate ortrifluoroacetate salt.

In one aspect, the present disclosure provides a method for treatingatherosclerosis in a mammalian subject, the method comprisingadministering an effective amount of (i) a peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable salt thereofand (ii) an antihyperlipidemic drug. In some embodiments, the peptideand the antihyperlipidemic drug are chemically linked. In someembodiments, the antihyperlipidemic drug includes one or more of:atorvastatin, simvastatin, pravastatin, fluvastatin, lovastatin,pitavastatin, rosuvastatin, clinofibrate, clofibrate, simfibrate,fenofibrate, bezafibrate, colestimide, colestyramine, ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin). In some embodiments, the antihyperlipidemicdrug is a statin. In some embodiments, the statin includes one or moreof: ADVICOR® (niacin extended-release/lovastatin), ALTOPREV® (lovastatinextended-release), CADUET® (amlodipine and atorvastatin), CRESTOR®(rosuvastatin), JUVISYNC® (sitagliptin/simvastatin), LESCOL®(fluvastatin), LESCOL XL (fluvastatin extended-release), LIPITOR®(atorvastatin), LIVALO® (pitavastatin), MEVACOR® (lovastatin),PRAVACHOL® (pravastatin), SIMCOR® (niacin extended-release/simvastatin),VYTORIN® (ezetimibe/simvastatin), and ZOCOR® (simvastatin).

In some embodiments, the peptide and the antihyperlipidemic agent areadministered simultaneously. In some embodiments, the peptide and theantihyperlipidemic agent are administered sequentially in either order.

In one aspect, the present disclosure provides a method for preventingatherosclerosis in a mammalian subject, the method comprisingadministering a therapeutically effective amount of a peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof.

In one aspect, the present disclosure provides a method for preventingatherosclerosis in a mammalian subject, the method comprisingadministering an effective amount of (i) a peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable salt thereofand (ii) an antihyperlipidemic drug. In some embodiments, the peptideand the antihyperlipidemic drug are chemically linked. In someembodiments, the antihyperlipidemic drug includes one or more of:atorvastatin, simvastatin, pravastatin, fluvastatin, lovastatin,pitavastatin, rosuvastatin, clinofibrate, clofibrate, simfibrate,fenofibrate, bezafibrate, colestimide, colestyramine, ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin). In some embodiments, the antihyperlipidemicdrug is a statin. In some embodiments, the statin includes one or moreof: ADVICOR® (niacin extended-release/lovastatin), ALTOPREV® (lovastatinextended-release), CADUET® (amlodipine and atorvastatin), CRESTOR®(rosuvastatin), JUVISYNC® (sitagliptin/simvastatin), LESCOL®(fluvastatin), LESCOL XL (fluvastatin extended-release), LIPITOR®(atorvastatin), LIVALO® (pitavastatin), MEVACOR® (lovastatin),PRAVACHOL® (pravastatin), SIMCOR® (niacin extended-release/simvastatin),VYTORIN® (ezetimibe/simvastatin), and ZOCOR® (simvastatin).

In some embodiments, the peptide and the antihyperlipidemic agent areadministered simultaneously. In some embodiments, the peptide and theantihyperlipidemic agent are administered sequentially in either order.In some embodiments, the subject is predisposed to atherosclerosis.

In one aspect, the present disclosure provides a method for amelioratingthe signs, symptoms or complications of atherosclerosis, the methodcomprising administering a therapeutically effective amount of a peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof.

In one aspect, the present disclosure provides a method for amelioratingthe signs, symptoms or complications of atherosclerosis, the methodcomprising administering an effective amount of (i) a peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable salt thereofand (ii) an antihyperlipidemic drug. In some embodiments, theantihyperlipidemic drug and the peptide are chemically linked. In someembodiments, the antihyperlipidemic drug includes one or more of:atorvastatin, simvastatin, pravastatin, fluvastatin, lovastatin,pitavastatin, rosuvastatin, clinofibrate, clofibrate, simfibrate,fenofibrate, bezafibrate, colestimide, colestyramine, ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin). In some embodiments, the antihyperlipidemicdrug is a statin. In some embodiments, the statin is includes one ormore of: ADVICOR® (niacin extended-release/lovastatin), ALTOPREV®(lovastatin extended-release), CADUET® (amlodipine and atorvastatin),CRESTOR® (rosuvastatin), JUVISYNC® (sitagliptin/simvastatin), LESCOL®(fluvastatin), LESCOL XL (fluvastatin extended-release), LIPITOR®(atorvastatin), LIVALO® (pitavastatin), MEVACOR® (lovastatin),PRAVACHO1® (pravastatin), SIMCOR® (niacin extended-release/simvastatin),VYTORIN® (ezetimibe/simvastatin), and ZOCOR® (simvastatin).

In some embodiments, treatment of atherosclerosis includes decreasingthe size or number of atherosclerotic plaques in the subject, and/ordecreasing the cholesterol content of an atherosclerotic plaque in thesubject.

In some embodiments, the peptide and the antihyperlipidemic agent areadministered simultaneously. In some embodiments, the peptide and theantihyperlipidemic agent are administered sequentially in either order.

In some embodiments, the signs, symptoms or complications ofatherosclerosis include one or more of: elevated levels totalcholesterol, very low density lipoprotein cholesterol (VLDL-C), lowdensity lipoprotein cholesterol (LDL-C), free (unesterified)cholesterol, cholesterol ester, phospholipids, triglycerides, andatherosclerotic lesions.

In one aspect, a method for delaying onset, ameliorating or eliminatingstatin side effects in a subject in need thereof is provided. In someembodiments, the method includes administering an effective amount of apeptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof, wherein the peptide is chemically linked to the statin. In someembodiments, the statin side effect includes one or more of myopathy,rhabdomyolysis kidney failure, diabetes, memory loss, decreased coenzymeQ10 levels and mitochondrial dysfunction. In some embodiments, thestatin includes one or more of atorvastatin, simvastatin, pravastatin,fluvastatin, lovastatin, pitavastatin, rosuvastatin, ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin).

In some aspects, a method for increasing statin dosage in a subject inneed thereof is provided. In some embodiments, the method includesadministering an effective amount of a statin at a first dosage level,and an aromatic-cationic peptide chemically linked to the statin;evaluating the subject for side-effects characteristic of the statin,wherein the side effects in the subject are reduced or absent ascompared to a control subject administered the statin and not thearomatic-cationic peptide; administering a statin at a second dosagelevel, wherein the second dosage level is higher than the first statindosage level. In some embodiments, the peptide isD-Arg-2′6′-Dmt-Lys-Phe-NH₂. In some embodiments, the statin includesLIPITOR® or CRESTOR®. In some embodiments, the side effectcharacteristic of the statin includes one or more of myopathy,rhabdomyolysis, kidney failure, diabetes, memory loss, decreasedcoenzyme Q10 levels and mitochondrial dysfunction.

In some embodiments, ameliorating the signs, symptoms or complicationsof atherosclerosis includes decreasing the size or number ofatherosclerotic plaques in the subject, and/or decreasing thecholesterol content of an atherosclerotic plaque in the subject.

As noted above, in some embodiments of the present methods andcompositions, the aromatic-cationic peptides of the present disclosure,such as D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof such as acetate or trifluoroacetate salt, and theantihyperlipidemic drug are chemically linked.

In some embodiments, the antihyperlipidemic drug is a statin. In someembodiments, the aromatic-cationic peptide and the statin are linkedusing a labile linkage that is hydrolyzed in vivo to release the peptideand the antihyperlipidemic drug. In some embodiments, the labile linkagecomprises an ester linkage, a carbonate linkage, or a carbamate linkage.

In some embodiments of the methods and compositions disclosed herein,the antihyperlipidemic drug is an anti-PCSK9 antibody. In someembodiments, the aromatic-cationic peptide and the anti-PCSK9 antibodyare linked using a bifunctional protein coupling agent. In someembodiments, the bifunctional protein coupling agent isN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), a bifunctional derivative of an imidoester, anactive ester, an aldehyde, a bis-azido compound, a bis-diazoniumderivative, a diisocyanate, or a bis-active fluorine compound. In someembodiments, the aromatic-cationic peptide and the anti-PCSK9 antibodyare linked using a labile linkage. In some embodiments, the labilelinkage comprises an ester linkage, a carbonate linkage, or a carbamatelinkage.

In some embodiments of the methods and compositions disclosed herein,the antihyperlipidemic agent is an Apo-B antisense oligonucleotide. Insome embodiments, the aromatic-cationic peptide and the Apo-B antisenseoligonucleotide are linked using a labile linkage. In some embodiments,the labile linkage comprises an ester linkage, a carbonate linkage, or acarbamate linkage.

In some embodiments, the peptides used in the compositions and methodsdisclosed herein is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In some embodiments, R¹ and R² are hydrogen; R³ and R⁴ are methyl; R⁵,R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

In some embodiments, the peptides used in the methods and compositionsdisclosed herein are defined by formula II:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In some embodiments, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, andR¹² are all hydrogen; and n is 4. In another embodiment, R¹, R², R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² are methyl; R¹⁰is hydroxyl; and n is 4.

In some embodiments, the peptide comprises a tyrosine or a2′,6′-dimethyltyrosine (Dmt) residue at the N-terminus. For example, thepeptide may have the formula Tyr-D-Arg-Phe-Lys-NH₂ or2′6′-Dmt-D-Arg-Phe-Lys-NH₂. In another embodiment, the peptide comprisesa phenylalanine or a 2′6′-dimethylphenylalanine residue at theN-terminus. For example, the peptide may have the formulaPhe-D-Arg-Phe-Lys-NH₂ or 2′6′-Dmp-D-Arg-Phe-Lys-NH₂. In a particularembodiment, the aromatic-cationic peptide has the formulaD-Arg-2′6′-Dmt-Lys-Phe-NH₂.

The aromatic-cationic peptides may be administered in a variety of ways.In some embodiments, the peptides may be administered orally, topically,intranasally, intraperitoneally, intravenously, subcutaneously, ortransdermally (e.g., by iontophoresis).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the % lesion area in control mice (vehicleonly—white bar) and test mice (aromatic-cationic peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂—dark bars) after 12 weeks of vehicle orpeptide administration. FIG. 1B is a photograph showing atheroscleroticlesions on aorta from a subject receiving vehicle only (left panel) oraromatic-cationic peptide (right panel).

FIG. 2 is a graph showing the thoracic aorta cholesterol (m/mg protein)in control mice (vehicle only—white bars) and test mice(aromatic-cationic peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂—dark bars) after12 weeks of vehicle or peptide administration. TC=total cholesterol;FC=free cholesterol; CE=cholesterol ester.

FIG. 3 is a graph showing mean leasion area (mm²) in control mice(vehicle only) and test mice (aromatic-cationic peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂) after 12 weeks.

FIG. 4A-H show levels of (A) total cholesterol; (B) free cholesterol;(C) cholesterol ester; (D) HDL-C; (E) VLDL-C; (F) LDL-C; (G)triglycerides; and (H) phospholipids after 0, 4, 8 and 12 weeks oftreatment with vehicle or peptide administration. Light bars in eachpanel represent data from control mice (vehicle only); dark barsrepresent data from the test mice (aromatic-cationic peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂).

FIG. 5 is a graph showing the effect of the aromatic-cationic peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ on coenzyme Q10 levels in fibroblast cells.The first bar represents saline treated fibroblasts; the second barrepresents fibroblasts treated with 10 nM peptide for 16-24 hours; thethird bar represents fibroblasts treated with 10 nM peptide for 5 days.

FIG. 6A is a diagram of the chemical structure of atorvastatin. FIG. 6Bis a diagram of the chemical structure of rosuvastatin.

FIG. 7A is a schematic diagram illustrating linkage of the peptideD-Arg-2′6′-Dmt -Lys-Phe-NH₂ to statins using a labile bond such that invivo hydrolysis of the pro-drug releases the two pharmaceutically activeagents. FIG. 7B is a schematic diagram illustrating linkage of thepeptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ to a hypolipidemic drug using alabile bond such that in vivo hydrolysis of the pro-drug releases thetwo pharmaceutically active agents.

FIGS. 8A and 8B show illustrative embodiments where when X═O, acarbonate-linked pro-drug is formed.

FIG. 9 is a diagram showing D-Arg-2′6′-Dmt-Lys-Phe-NH₂ and statinpotential reactive linking sites for pro-drug formation (arrows).

FIG. 10 shows illustrative pro-drugs in which theD-Arg-2′6′-Dmt-Lys-Phe-NH₂ peptide is linked to CRESTOR® or LIPITOR®using a carbonate linkage.

FIG. 11 shows illustrative pro-drugs in which theD-Arg-2′6′-Dmt-Lys-Phe-NH₂ peptide is linked to CRESTOR® using acarbamate linkage.

FIG. 12 shows exemplary self immolating moieties.

FIGS. 13A and 13B show exemplary schematics for formulations linking anaromatic-cationic peptide to a statin (A); and linking anaromatic-cationic peptide to an antibody (B). FIGS. 13C and 13D showexemplary schematics for formulations linking an aromatic-cationicpeptide to a antihyperlipidemic drug (C); and linking anaromatic-cationic peptide to an antibody (D).

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention.

In practicing the present invention, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively.

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of, or a decrease in,atherosclerosis or one or more symptoms associated with atherosclerosis.In the context of therapeutic or prophylactic applications, the amountof a composition administered to the subject will depend on the type andseverity of the disease and on the characteristics of the individual,such as general health, age, sex, body weight and tolerance to drugs. Itwill also depend on the degree, severity and type of disease. Theskilled artisan will be able to determine appropriate dosages dependingon these and other factors. The compositions can also be administered incombination with one or more additional therapeutic compounds. In themethods described herein, the aromatic-cationic peptides may beadministered to a subject having one or more signs or symptoms ofatherosclerosis. For example, a “therapeutically effective amount” ofthe aromatic-cationic peptides is meant levels in which thephysiological effects of atherosclerosis are, at a minimum, ameliorated.In some embodiments, signs, symptoms or complications of atherosclerosisinclude, but are not limited to: increased plasma total cholesterol,increased plasma free cholesterol, increased plasma cholesterol ester,lesions (e.g. aortic lesions), increased plasma very low-densitylipoprotein, increased plasma low density lipoprotein and/or increasedplasma phospholipids.

An “isolated” or “purified” polypeptide or peptide is substantially freeof cellular material or other contaminating polypeptides from the cellor tissue source from which the agent is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.For example, an isolated aromatic-cationic peptide would be free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe agent. Such interfering materials may include enzymes, hormones andother proteinaceous and nonproteinaceous solutes.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art.

As used herein, the term “simultaneous” therapeutic use refers to theadministration of at least two active ingredients by the same route andat the same time or at substantially the same time.

As used herein, the term “separate” therapeutic use refers to anadministration of at least two active ingredients at the same time or atsubstantially the same time by different routes.

As used herein, the term “sequential” therapeutic use refers toadministration of at least two active ingredients at different times,the administration route being identical or different. Moreparticularly, sequential use refers to the whole administration of oneof the active ingredients before administration of the other or otherscommences. It is thus possible to administer one of the activeingredients over several minutes, hours, or days before administeringthe other active ingredient or ingredients. There is no simultaneoustreatment in this case.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. For example, a subject issuccessfully “treated” for atherosclerosis if, after receiving atherapeutic amount of the aromatic-cationic peptides according to themethods described herein, the subject shows observable and/or measurablereduction in or absence of one or more signs, symptoms or complicationsof atherosclerosis, such as, e.g., reduced total plasma cholesterol,free cholesterol, cholesterol ester, very low-density lipoproteincholesterol (VLDL-C), low density lipoprotein cholesterol (LDL-C),phospholipids, lesion size and/or number (e.g. aortic lesions), loweredlevels of cholesterol in the aortic tissue and/or aortic plaques, and/orlowered levels of cholesterol atherosclerotic lesions or plaques ascompared to a subject not treated with the therapeutic aromatic-cationicpeptide. It is also to be appreciated that the various modes oftreatment or prevention of medical conditions as described are intendedto mean “substantial,” which includes total but also less than totaltreatment or prevention, and wherein some biologically or medicallyrelevant result is achieved.

As used herein, “prevention” or “preventing” of a disorder or conditionrefers to one or more compounds that, in a statistical sample, reducesthe occurrence of the disorder or condition in the treated samplerelative to an untreated control sample, or delays the onset or reducesthe severity of one or more symptoms of the disorder or conditionrelative to the untreated control sample. As used herein, preventingatherosclerosis includes reducing to, or maintaining at, normal levelsone or more signs, symptoms or complications of atherosclerosisincluding, but not limited to total plasma cholesterol, freecholesterol, cholesterol ester, very low-density lipoproteincholesterol, low density lipoprotein cholesterol, phospholipids, lesionsize and/or number (e.g. aortic lesions), levels of cholesterol in theaortic tissue and/or aortic plaques, and/or levels of cholesterolatherosclerotic lesions or plaques as compared to a subject not treatedwith the therapeutic aromatic-cationic peptide.

As used herein, the terms “drug” and “agent” are synonymous.

As used herein the terms “antihyperlipidemic agent” or“antihyperlipidemic drug” are synonymous with the terms “hypolipidemicagent” or “hypolipidemic drug.”

Methods of Prevention or Treatment

The present technology relates to the treatment or prevention ofatherosclerosis by administration of aromatic-cationic peptides and, insome embodiments, aromatic-cationic peptides in conjunction with one ormore active agents to a subject in need thereof. For example, thepresent technology relates to the treatment or prevention ofatherosclerosis by administration of aromatic-cationic peptides, and insome embodiments, an aromatic-cationic peptide and one or moreantihyperlipidemic drugs (e.g., statins) to a subject in need thereof.In some embodiments, the antihyperlipidemic drug and thearomatic-cationic peptide are chemically linked.

In one embodiment, the aromatic-cationic peptides and/or one or moreagents are administered in dosages that are sub-therapeutic for eachagent when administered separately. However, in some embodiments, thecombination of the two agents results in synergism, which provides anenhanced effect that is not observed when each of the agents areadministered individually at higher doses. In one embodiment, theadministration of the aromatic-cationic peptide and one or more agents“primes” the tissue, so that it is more responsive to the therapeuticeffects of the other agent. Thus, in some embodiments, a lower dose ofthe aromatic-cationic peptide and/or the one or more agents (e.g.,antihyperlipidemic drugs, such as statins) can be administered, and yet,a therapeutic effect is still observed.

In some embodiments, the subject (e.g, a subject suffering fromatherosclerosis, and/or exhibiting the signs, symptoms or complicationsof atherosclerosis, and/or who is predisposed to atherosclerosis or thesigns, symptoms or complications of atherosclerosis) is administered thepeptide, or is administered a peptide and one or more antihyperlipidemicdrugs (e.g., statins) simultaneously, separately, or sequentially. Insome embodiments, the subject is administered the peptide or isadministered the peptide and one or more antihyperlipidemic drugs (e.g.,statins), before atherosclerosis or before the signs, symptoms orcomplications of atherosclerosis are evident.

Aromatic-cationic peptides are water-soluble and highly polar. Despitethese properties, the peptides can readily penetrate cell membranes. Thearomatic-cationic peptides typically include a minimum of three aminoacids or a minimum of four amino acids, covalently joined by peptidebonds. The maximum number of amino acids present in thearomatic-cationic peptides is about twenty amino acids covalently joinedby peptide bonds. Suitably, the maximum number of amino acids is abouttwelve, more preferably about nine, and most preferably about six.

The amino acids of the aromatic-cationic peptides can be any amino acid.As used herein, the term “amino acid” is used to refer to any organicmolecule that contains at least one amino group and at least onecarboxyl group. Typically, at least one amino group is at the a positionrelative to a carboxyl group. The amino acids may be naturallyoccurring. Naturally occurring amino acids include, for example, thetwenty most common levorotatory (L) amino acids normally found inmammalian proteins, i.e., alanine (Ala), arginine (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),and valine (Val). Other naturally occurring amino acids include, forexample, amino acids that are synthesized in metabolic processes notassociated with protein synthesis. For example, the amino acidsornithine and citrulline are synthesized in mammalian metabolism duringthe production of urea. Another example of a naturally occurring aminoacid includes hydroxyproline (Hyp).

The peptides optionally contain one or more non-naturally occurringamino acids. For example, the peptide may have no amino acids that arenaturally occurring. The non-naturally occurring amino acids may belevorotary (L-), dextrorotatory (D-), or mixtures thereof. Non-naturallyoccurring amino acids are those amino acids that typically are notsynthesized in normal metabolic processes in living organisms, and donot naturally occur in proteins. In addition, the non-naturallyoccurring amino acids suitably are also not recognized by commonproteases. The non-naturally occurring amino acid can be present at anyposition in the peptide. For example, the non-naturally occurring aminoacid can be at the N-terminus, the C-terminus, or at any positionbetween the N-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include α-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide is thederivatization of a carboxyl group of an aspartic acid or a glutamicacid residue of the peptide. One example of derivatization is amidationwith ammonia or with a primary or secondary amine, e.g. methylamine,ethylamine, dimethylamine or diethylamine. Another example ofderivatization includes esterification with, for example, methyl orethyl alcohol. Another such modification includes derivatization of anamino group of a lysine, arginine, or histidine residue. For example,such amino groups can be acylated. Some suitable acyl groups include,for example, a benzoyl group or an alkanoyl group comprising any of theC₁-C₄ alkyl groups mentioned above, such as an acetyl or propionylgroup.

The non-naturally occurring amino acids are preferably resistant, andmore preferably insensitive, to common proteases. Examples ofnon-naturally occurring amino acids that are resistant or insensitive toproteases include the dextrorotatory (D-) form of any of theabove-mentioned naturally occurring L-amino acids, as well as L- and/orD-non-naturally occurring amino acids. The D-amino acids do not normallyoccur in proteins, although they are found in certain peptideantibiotics that are synthesized by means other than the normalribosomal protein synthetic machinery of the cell. As used herein, theD-amino acids are considered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides should have lessthan five, preferably less than four, more preferably less than three,and most preferably, less than two contiguous L-amino acids recognizedby common proteases, irrespective of whether the amino acids arenaturally or non-naturally occurring. Optimally, the peptide has onlyD-amino acids, and no L-amino acids. If the peptide contains proteasesensitive sequences of amino acids, at least one of the amino acids ispreferably a non-naturally-occurring D-amino acid, thereby conferringprotease resistance. An example of a protease sensitive sequenceincludes two or more contiguous basic amino acids that are readilycleaved by common proteases, such as endopeptidases and trypsin.Examples of basic amino acids include arginine, lysine and histidine.

The aromatic-cationic peptides should have a minimum number of netpositive charges at physiological pH in comparison to the total numberof amino acid residues in the peptide. The minimum number of netpositive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r). The minimum number of net positive chargesdiscussed below are all at physiological pH. The term “physiological pH”as used herein refers to the normal pH in the cells of the tissues andorgans of the mammalian body. For instance, the physiological pH of ahuman is normally approximately 7.4, but normal physiological pH inmammals may be any pH from about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid. Typically, a peptide has apositively charged N-terminal amino group and a negatively chargedC-terminal carboxyl group. The charges cancel each other out atphysiological pH.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1. In thisembodiment, the relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) is asfollows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≦ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≦ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, suitably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a). Naturally occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides should also have a relationship betweenthe minimum number of aromatic groups (a) and the total number of netpositive charges at physiological pH (p_(t)) wherein 3a is the largestnumber that is less than or equal to p_(t)+1, except that when p_(t) is1, a may also be 1. In this embodiment, the relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (p_(t)) is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≦ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (2a ≦ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal. In one embodiment, thearomatic-cationic peptide is a tripeptide having two net positivecharges and at least one aromatic amino acid. In a particularembodiment, the aromatic-cationic peptide is a tripeptide having two netpositive charges and two aromatic amino acids.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are suitably amidated with, for example, ammonia to form theC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group. The free carboxylate groups of theasparagine, glutamine, aspartic acid, and glutamic acid residues notoccurring at the C-terminus of the aromatic-cationic peptides may alsobe amidated wherever they occur within the peptide. The amidation atthese internal positions may be with ammonia or any of the primary orsecondary amines described above.

Aromatic-cationic peptides include, but are not limited to, thefollowing peptide examples:

Lys-D-Arg-Tyr-NH₂ Phe-D-Arg-His D-Tyr-Trp-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂Tyr-His-D-Gly-Met Phe-Arg-D-His-Asp Tyr-D-Arg-Phe-Lys-Glu-NH₂Met-Tyr-D-Lys-Phe-Arg D-His-Glu-Lys-Tyr-D-Phe-ArgLys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂ Phe-D-Arg-Lys-Trp-Tyr-D-Arg-HisGly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysLys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysAsp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheTyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-PhePhe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-ThrTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-LysGlu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH₂Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-GlyD-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-PheHis-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-AspThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂

In one embodiment, the aromatic-cationic peptide has the formulaPhe-D-Arg-Phe-Lys-NH₂. In another embodiment, the aromatic-cationicpeptide has the formula D-Arg-2′6′-Dmt-Lys-Phe-NH₂.

The peptides mentioned herein and their derivatives can further includefunctional analogs. A peptide is considered a functional analog if theanalog has the same function as the stated peptide. The analog may, forexample, be a substitution variant of a peptide, wherein one or moreamino acids are substituted by another amino acid. Suitable substitutionvariants of the peptides include conservative amino acid substitutions.Amino acids may be grouped according to their physicochemicalcharacteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group is generally more likely to alter thecharacteristics of the original peptide.

Examples of peptides include, but are not limited to, thearomatic-cationic peptides shown in Table 5.

TABLE 5 Peptide Analogs with Mu-Opioid Activity Amino Acid Amino AcidAmino Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ TyrD-Arg Phe Dab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂2′6′Dmt D-Arg Phe Lys-NH(CH₂)₂—NH-dns NH₂ 2′6′Dmt D-Arg PheLys-NH(CH₂)₂—NH-atn NH₂ 2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-Cit PheLys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg Phe Orn NH₂ 2′6′DmtD-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg PheAhp(2-aminoheptanoic acid) NH₂ Bio-2′6′Dmt D-Arg Phe Lys NH₂ 3′5′DmtD-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′Dmt D-Arg Phe Dab NH₂3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ Tyr D-Arg Tyr Orn NH₂Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg Tyr Lys NH₂2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′Dmt D-Arg TyrDap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg 2′6′Dmt Orn NH₂2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dap NH₂ 3′5′DmtD-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′Dmt D-Arg3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ Tyr D-Lys Phe Dap NH₂ TyrD-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂ 2′6′DmtD-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys Phe Arg NH₂2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′Dmt D-Lys PheDab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ Tyr D-LysTyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr Dab NH₂ Tyr D-Lys TyrDap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys Tyr Orn NH₂ 2′6′DmtD-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys 2′6′Dmt LysNH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dab NH₂ 2′6′DmtD-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDap NH₂ 2′6′Dmt D-Arg PheatnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′Dmt D-Lys 3′5′Dmt Orn NH₂3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dap NH₂ Tyr D-LysPhe Arg NH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-Dab Phe Arg NH₂ Tyr D-Dap PheArg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′DmtD-Orn Phe Arg NH₂ 2′6′Dmt D-Dab Phe Arg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂3′5′Dmt D-Arg Phe Arg NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ 3′5′Dmt D-Orn PheArg NH₂ Tyr D-Lys Tyr Arg NH₂ Tyr D-Orn Tyr Arg NH₂ Tyr D-Dab Tyr ArgNH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′Dmt D-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn 2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt ArgNH₂ 3′5′Dmt D-Dap 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′DmtD-Lys 3′5′Dmt Arg NH₂ 3′5′Dmt D-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe LysNH₂ Mmt D-Arg Phe Orn NH₂ Mmt D-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂Tmt D-Arg Phe Lys NH₂ Tmt D-Arg Phe Orn NH₂ Tmt D-Arg Phe Dab NH₂ TmtD-Arg Phe Dap NH₂ Hmt D-Arg Phe Lys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-ArgPhe Dab NH₂ Hmt D-Arg Phe Dap NH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys PheOrn NH₂ Mmt D-Lys Phe Dab NH₂ Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe ArgNH₂ Tmt D-Lys Phe Lys NH₂ Tmt D-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂Tmt D-Lys Phe Dap NH₂ Tmt D-Lys Phe Arg NH₂ Hmt D-Lys Phe Lys NH₂ HmtD-Lys Phe Orn NH₂ Hmt D-Lys Phe Dab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-LysPhe Arg NH₂ Mmt D-Lys Phe Arg NH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab PheArg NH₂ Mmt D-Dap Phe Arg NH₂ Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe ArgNH₂ Tmt D-Orn Phe Arg NH₂ Tmt D-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂Tmt D-Arg Phe Arg NH₂ Hmt D-Lys Phe Arg NH₂ Hmt D-Orn Phe Arg NH₂ HmtD-Dab Phe Arg NH₂ Hmt D-Dap Phe Arg NH₂ Hmt D-Arg Phe Arg NH₂ Dab =diaminobutyric Dap = diaminopropionic acid Dmt = dimethyltyrosine Mmt =2′-methyltyrosine Tmt = N,2′,6′-trimethyltyrosine Hmt =2′-hydroxy,6′-methyltyrosine dnsDap = β-dansyl-L-α,β-diaminopropionicacid atnDap = β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of peptides also include, but are not limited to, thearomatic-cationic peptides shown in Table 6.

TABLE 6 Peptide Analogs Lacking Mu-Opioid Activity Amino Acid Amino AcidAmino Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-ArgLys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-ArgPhe Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTrp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg TrpDmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg PheLys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl alanine

The amino acids of the peptides shown in Table 5 and 6 may be in eitherthe L- or the D-configuration.

In some embodiments, the aromatic-cationic peptide is a peptide having:

at least one net positive charge;

-   -   a minimum of four amino acids;    -   a maximum of about twenty amino acids;    -   a relationship between the minimum number of net positive        charges (p_(m)) and the total number of amino acid residues (r)        wherein 3p_(m) is the largest number that is less than or equal        to r+1; and a relationship between the minimum number of        aromatic groups (a) and the total number of net positive charges        (p_(t)) wherein 2a is the largest number that is less than or        equal to p_(t)+1, except that when a is 1, p_(t) may also be 1.

In one embodiment, 2p_(m) is the largest number that is less than orequal to r+1, and a may be equal to p_(t). The aromatic-cationic peptidemay be a water-soluble peptide having a minimum of two or a minimum ofthree positive charges.

In one embodiment, the peptide comprises one or more non-naturallyoccurring amino acids, for example, one or more D-amino acids. In someembodiments, the C-terminal carboxyl group of the amino acid at theC-terminus is amidated. In certain embodiments, the peptide has aminimum of four amino acids. The peptide may have a maximum of about 6,a maximum of about 9, or a maximum of about 12 amino acids.

In one embodiment, the peptide comprises a tyrosine or a2′6′-dimethyltyrosine (Dmt) residue at the N-terminus. For example, thepeptide may have the formula Tyr-D-Arg-Phe-Lys-NH₂ or2′6′-Dmt-D-Arg-Phe-Lys-NH₂. In another embodiment, the peptide comprisesa phenylalanine or a 2′6′-dimethylphenylalanine residue at theN-terminus. For example, the peptide may have the formulaPhe-D-Arg-Phe-Lys-NH₂ or 2′6′-Dmp-D-Arg-Phe-Lys-NH₂. In a particularembodiment, the aromatic-cationic peptide has the formulaD-Arg-2′6′-Dmt-Lys-Phe-NH₂.

In one embodiment, the peptide is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

In one embodiment, the peptide is defined by formula II:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, and R¹² are all hydrogen; and n is 4. In another embodiment, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² aremethyl; R¹⁰ is hydroxyl; and n is 4.

The peptides may be synthesized by any of the methods well known in theart. Suitable methods for chemically synthesizing the protein include,for example, those described by Stuart and Young in Solid Phase PeptideSynthesis, Second Edition, Pierce Chemical Company (1984), and inMethods Enzymol., 289, Academic Press, Inc, New York (1997).

Active Agents for Use in Combination Therapy with Aromatic-CationicPeptides

In some aspects, the methods disclosed herein provide combinationtherapies for the treatment of atherosclerosis, and/or statin-relatedside effects comprising administering an effective amount of anaromatic-cationic peptide or a pharmaceutically acceptable salt thereof,such as acetate or trifluoroacetate salt, in combination with one ormore active agents (therapeutic agents/active ingredients). Thus, forexample, the combination of active ingredients may be: (1) co-formulatedand administered or delivered simultaneously in a combined formulation;(2) delivered by alternation or in parallel as separate formulations; or(3) by any other combination therapy regimen known in the art. Whendelivered in alternation therapy, the methods described herein maycomprise administering or delivering the active ingredientssequentially, e.g., in separate solution, emulsion, suspension, tablets,pills or capsules, or by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e., serially, whereas insimultaneous therapy, effective dosages of two or more activeingredients are administered together. Various sequences of intermittentcombination therapy may also be used.

In some embodiments, the combination therapy comprises administering toa subject in need thereof an aromatic-cationic peptide compositioncombined with one or more active agents, e.g., one or moreantihyperlipidemic agent (e.g., a statin). In some embodiments, theantihyperlipidemic drug and the aromatic-cationic peptide are chemicallylinked.

Antihyperlipidemic (Hypolipidemic) Drugs and Statins

In some embodiments, the one or more additional active agentsadministered with one or more aromatic-cationic peptides disclosedherein (such as D-Arg-2′6′-Dmt-Lys-Phe-NH₂,) or a pharmaceuticallyacceptable salt thereof, such as acetate or trifluoroacetate salt, is anantihyperlipidemic (hypolipidemic) drug. As used herein, the terms“antihyperlipidemic” and “hypolipedimic” are synonymous and are usedinterchangeably. For example, in some embodiments, the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt is administeredsimultaneous to the antihyperlipidemic agent (drug). In someembodiments, the antihyperlipidemic drug and the aromatic-cationicpeptide are chemically linked. In some embodiments, the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt is administered priorto or subsequent to the antihyperlipidemic agent (drug).

In some embodiments, the antihyperlipidemic drug comprises one or morestatins. In some embodiments, the statin is a combination drugcomprising a statin and a non-statin. Exemplary, non-limiting statinsinclude one or more of the following: lovastatin, (e.g., ADVICOR®(niacin extended-release/lovastatin), ALTOPREV™ (lovastatinextended-release), MEVACOR®), atorvastatin, (e.g., CADUET® (amlodipineand atorvastatin), LIPITOR®), rosuvastatin and/or rosuvastatin calcium,(e.g., CRESTOR®), simvastatin, (e.g., JUVISYNC®(sitagliptin/simvastatin), SIMCOR® (niacinextended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin) andZOCOR®), fluvastatin and/or fluvastatin sodium, (e.g., LESCOL®, LESCOLXL (fluvastatin extended-release)), pitavastatin (e.g., LIVALO®),pravastatin and/or pravastatin sodium (e.g., PRAVACHOL®)

In some embodiments, the hypolipidemic agent is a lipid lowering drug.In some embodiments, the active agent is an LDL lowering drug. In someembodiments, the active agent is a triglyceride lowering drug. In someembodiments, the active agent is an HDL elevating drug.

In some embodiments, the hypolipidemic agent is a cholesteryl estertransfer protein (CETP) inhibitor. In some embodiments, the CETPinhibitor is TORCETRAPIB®, ANACETRAPIB, or DALCETRAPIB®.

In some embodiments, the hypolipidemic agent targets proproteinconvertase subtilisin/kexin type 9 (PCSK9). In some embodiments, theagent is a PCSK9 inhibitor. In some embodiments, the agent inhibitsPCSK9 function. In some embodiments, the agent inhibits PCSK9expression. In some embodiments, the PCSK9 inhibitor targets PCSK9 mRNA.In some embodiments, the PCSK9 inhibitor is a PCSK9 siRNA. In someembodiments, the PCSK9 inhibitor is ALN-PCS or REGN727. In someembodiments, the one ore more therapies targeting PCSK9 is an anti-PCSK9antibody. In some embodiments, the anti-PCSK9 antibody is a monoclonalantibody. In some embodiments, the anti-PCSK9 antibody is humanized. Insome embodiments, the anti-PCSK9 antibody is a human antibody. In someembodiments, at least a portion of the framework sequence of theanti-PCSK9 antibody is a human consensus framework sequence. In someembodiments, the antibody is an antibody fragment selected from a Fab,Fab′-SH, Fv, scFv, or Fab₂ fragment.

In some embodiments, the hypolipidemic agent is a fibrate. In someembodiments, the fibrate is LIPOFEN® (fenofibrate), LOPID®(gemfibrozil), TRICOR® (fenofibrate), LOFIBRA® (fenofibrate), ATROMID-S®(clofibrate), TRILIPIX® (fenofibric acid), FENOGLIDE® (fenofibrate),ANTARA® (fenofibrate), FIBRICOR® (fenofibric acid), or TRIGLIDE®(fenofibrate). In some embodiments, the hypolipidemic agent isclinofibrate, simfibrate, benzafibrate.

In some embodiments, the hypolipidemic agent is niacin. In someembodiments, the niacin is NIASPAN®. In some embodiments, the niacin isNIACOR®.

In some embodiments, the hypolipidemic agent is a bile acid resin. Insome embodiments, the bile acid resin is QUESTRAN®, QUESTRAN LIGHT®,COLESTID, or WELCHOL®.

In some embodiments, the hypolipidemic agent prevents the absorption ofdietary lipids. In some embodiments the agent is ezetimibe (e.g.,ZETIA®), orlistat (e.g., XENICAL®), or a phytosterol.

In some embodiments, the hypolipidemic agent is a squalene synthaseinhibitor. In some embodiments, the hypolipidemic agent is ApoA-1MILANO®. In some embodiments, the hypolipidemic agent is AGI-1067. Insome embodiments, the hypolipidemic agent is MIPOMERSEN®.

In some embodiments, the hypolipidemic agent is one or more ofcolestimide and colestyramine.

In some embodiments, the additional active agent administered incombination with an aromatic-cationic peptide of the present disclosurecomprises an agent effective for apolipoprotein therapy. For example, inone aspect, the present disclosure provides combination therapiescomprising administering an effective amount of peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable salt, suchas acetate or trifluoroacetate salt in combination with one or moretherapies targeting apolipoprotein (Apo). In some embodiments, theanti-Apo therapy is an antisense therapy. In some embodiments, theanti-Apo therapy is an antisense therapy targeting apolipoprotein B(Apo-B). In some embodiments, the anti-Apo-B antisense therapy is anantisense oligonucleotide, for example, comprising nucleotides linkedwith phosphorothioate linkages. In some embodiments, the anti-Apo-Bantisense therapy is an antisense therapeutic that targets the messengerRNA for apolioprotein B MIPOMERSEN®. In some embodiments, the anti-Apo-Bantisense therapy is KYNAMRO®. In some embodiments, the anti-Apo-Bantisense therapy has the following sequence:G*-C*-C*-U*-C*-dA-dG-dT-dC-dT-dG-dmC-dT-dT-dmC-G*-C*-A*-C*-C*, whered=2′-deoxy, *=2′-O-(2-methoxyethyl), with 3′→5′ phosphorothioatelinkages. In some embodiments, the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ ora pharmaceutically acceptable salt thereof, such as acetate ortrifluoroacetate salt is administered simultaneous to the anti-Apo-Bagent. In some embodiments, the anti-Apo-B agent and thearomatic-cationic peptide are chemically linked. In some embodiments,the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptablesalt thereof, such as acetate or trifluoroacetate salt is administeredprior to or subsequent to the anti-Apo-B agent.

Statin Structure

As noted above, in some embodiments, the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt is administeredsimultaneous to the statin. In some embodiments, the statin and thearomatic-cationic peptide are chemically linked. In some embodiments,the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptablesalt, such as acetate or trifluoroacetate salt is administered prior toor subsequent to the statin. Typically, the structural components ofstatins include a dihydroxyheptanoic acid unit and a ring system withdifferent substituents. The statin pharmacophore is modifiedhydroxyglutaric acid component, which is structurally similar to theendogenous substrate HMG CoA and the mevaldyl CoA transition stateintermediate. The statin pharmacophore binds to the same active site asthe substrate HMG-CoA and inhibits the HMG-CoA reductase enzyme. TheHMG-CoA reductase enzyme is stereoselective and as a result functionalstatins typically have the 3R,5R stereochemistry.

Statins can be separated into two classes: type 1 (e.g. lovastatin,pravastatin, simvastatin) and type 2 (e.g. fluvastatin, cerivastatin,atorvastatin, rosuvastatin). Type 1 statins include a substituteddecalin-ring structure that resemble mevastatin, a compound isolatedfrom the mold Penicillium citrinum. Lovastatin was isolated from themold Aspergillus terreus and pravastatin and simvastatin are chemicallymodified versions of lovastatin. Type 2 statins are fully synthetic andhave larger substituent groups that interact with the HMG-CoA reductaseenzyme. In addition, type 2 statins substitute fluorophenyl group forthe butyryl group found on type 1 statins. The fluorophenyl groupprovides additional polar interactions typically resulting in tighterbinding with the HMG-CoA reductase enzyme. Rosuvastatin has asulfonamide group that is hydrophilic and increases binding affinitywith the HMG-CoA reductase enzyme.

Atorvastatin Structure

Atorvastatin is a ring-opened hydroxy-acid of trans-6-[2-(3- or4-carboxamido-substituted pyrrol-1-yl)alkyl]-4-hydroxypyran-2-one (seee.g., FIG. 6A for the chemical structure). The general structure ofatorvastatin and related compounds is provided in Formula I:

-   -   wherein X is —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂— or —CH₂CH(CH₃).

R₁ is 1-naphthyl; 2-naphthyl; cyclohexyl; norbornenyl; 2-, 3-, or4-pyridinyl; phenyl, phenyl substituted with fluorine, chlorine,bromine, hydroxyl; trifluoromethyl; alkyl of from one to four carbonatoms, alkoxy of from one to four carbon atoms, or alkanoyloxy of fromtwo to eight carbon atoms.

Either R₂ or R₃ is —CONR₅R₆ where R₅ and R₆ are independently hydrogen;alkyl of from one to six carbon atoms; 2-, 3-, or 4-pyridinyl; phenyl;phenyl substituted with fluorine, chlorine, bromine, cyano,trifluoromethyl, or carboalkoxy of from three to eight carbon atoms; andthe other of R₂ or R₃ is hydrogen; alkyl of from one to six carbonatoms; cyclopropyl; cyclobutyl; cyclopentyl; cyclohexyl; phenyl; orphenyl substituted with fluorine, chlorine, bromine, hydroxyl;trifluoromethyl; alkyl of from one to four carbon atoms, alkoxy of fromone to four carbon atoms, or alkanoyloxy of from two to eight carbonatoms.

R₄ is alkyl of from one to six carbon atoms; cyclopropyl; cyclobutyl;cyclopentyl; cyclohexyl; or trifluoromethyl.

Rosuvastatin Structure

Rosuvastatin is a compound related to the general structure set forth informula II:

wherein R₁ is lower alkyl, aryl or aralkyl, each of which may have oneor more substituents: R₂ and R₃ each is independently hydrogen, loweralkyl, or aryl, and each of said lower alkyl and aryl may have one ormore substituents; R₄ is hydrogen, lower alkyl, or a cation capable offorming a non-toxic pharmaceutically acceptable salt; X is sulfur,oxygen, or sulfonyl, or imino which may have a substituent; the dottedline represents the presence or absence of a double bond, or thecorresponding ring-closed lactone. (See e.g., FIG. 6B)

The term “lower alkyl” refers to a straight, branched, or cyclic C₁ toC₆ alkyl, including methyl, ethyl, n-propyl, isopropyl, cyclopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl,isopentyl, neopentyl, tert-pentyl, cyclopentyl, n-hexyl, and isohexyland the like. Further, the lower alkyl may be substituted by 1 to 3substituents independently selected from the group consisting ofhalogen, amino, and cyano. Halogen means fluorine, chlorine, bromine andiodine.

The term “aryl” refers to C₆ to C₁₂ aromatic group including phenyl,tolyl, xylyl, biphenyl, naphthyl, and the like. The aryl may have 1 to 3substituents independently selected from the group consisting of loweralkyl, halogen, amino, and cyano. Preferred aryl is phenyl substitutedby 1 to 3 halogens.

The term “aralkyl” refers to C₁ to C₆ lower alkyl substituted by C₆ toC₁₂ aromatic aryl group defined above. Examples of them are benzyl,phenethyl, phenylpropyl and the like, each of which may have 1 to 3substituents independently selected from the group consisting of loweralkyl halogen, amino, cyano, and the like.

The term “a cation capable of forming a non-toxic pharmaceuticallyacceptable salt” refers to alkali metal ion, alkaline earth metal ion,and ammonium ion. Examples of alkali metal are lithium, sodium,potassium, and cesium, and examples of alkaline earth metal areberyllium, magnesium, and calcium. Especially, sodium and calcium arepreferred.

Examples of “acyl” are formyl acetyl, propionyl, butyryl, isobutyryl,valeryl, and isovaleryl.

In the term “imino which may have a substituent,” preferred substituentsare acyl, optionally substituted amino, and substituted sulfonyl.

The term “substituted amino as substituent” means amino groupsubstituted by sulfonyl and alkylsulfonyl. Examples of them are sulfonylamino and methanesulfonyl amino.

The term “substituted sulfonyl as substituent” means sulfonyl groupsubstituted by alkyl, amino, or alkylamino. Examples of them aremethanesulfonyl, sulfamoyl, methylsulfamoyl, and N-methylsulfamoyl.

LIPITOR® and CRESTOR®

Atorvastatin (also known by the trademarked name LIPITOR®) can be usedto reduce the risk of myocardial infarction, stroke, revascularizationprocedures, and angina in patients without coronary heart disease, butwith multiple risk factors. Such risk factors include but are notlimited to age, smoking, hypertension, low HDL-C, or a family history ofearly coronary heart disease. Atorvastatin can also be used to reducethe risk of myocardial infarction and stroke in patients with type 2diabetes without coronary heart disease, but with multiple risk factors.Such risk factors include but are not limited to retinopathy,albuminuria, smoking, or hypertension. Atorvastatin can be used toreduce the risk of non-fatal MI, fatal and non-fatal stroke,revascularization procedures, hospitalization for coronary heartfailure, and angina in patients with coronary heart disease.Atorvastatin can be used to reduce elevated total cholesterol, LDL-C,ApoB, and triglyceride levels and increase HDL-C in adult patients withprimary hyperlipidemia (heterozygous familial and nonfamilal) and mixeddyslipidemia. Atorvastatin can be used to reduce elevated triglyceridesin patients with hypertriglyceridemia and primarydysbetalipoproteinemia. Atorvastatin can also be used to reduce totalcholesterol and LDL-C in patients with homozygous familialhypercholesterolemia (HoFH). Atorvastatin can be used to reducedelevated total cholesterol, LDL-C, and ApoB levels in boys andpostmenarchal girls between the ages of 10-17, with heterozygousfamilial hypercholesterolemia after failing an adequate trial of diettherapy.

Atorvastatin has also been used to treat spinal cord injury in rodents,promoting locomotion and tissue sparing, as well as reducinginflammation when administered both pre- and post-injury. In addition,atorvastatin has been utilized in an in vitro model of hepatitis C virus(HCV) infection (alone and with interferon). In such a system,atorvastatin (as well as lovastatin, simvastatin, fluvastatin, andpitavastatin) was shown to have an anti-HCV effect. Accordingly, statinsmay be suitable for concurrent therapy with interferon.

Rosuvastatin (also known by the trade name CRESTOR®) can be used totreat patients with primary hyperlipidemia and mixed dyslipidemia as anadjunct to diet to reduce levels of total cholesterol, LDL-C, ApoB,nonHDL-C, and triglyceride levels and to increase levels of HDL-C.Rosuvastatin can also be used to treat patients with:hypertriglyceridemia as an adjunct to diet, primarydysbetalipoproteinemia (Type II hyperlipoproteinemia) as an adjunct todiet, and homozygous familial hypercholesterolemia (HoFH). Rosuvastatincan be used to slow the progression of atherosclerosis in patients aspart of a treatment strategy to lower total cholesterol and LDL-C as anadjunct to diet. Rosuvastatin can be used to treat patients 10 to 17years old with heterozygous familial hypercholesterolemia (HeFH) toreduce elevated total cholesterol, LDL-C, and ApoB after failing anadequate trial of diet therapy. Rosuvastatin can be used for reducingthe risk of myocardial infarction, stroke, and arterialrevascularization procedures in patients without evident coronary heartdisease, but with multiple risk factors. Such risk factors includehypertension, low HDL-C, smoking, or a family history of prematurecoronary heart disease.

Therapeutic Uses of Aromatic-Cationic Peptides and Active Agents

Atherosclerosis

The aromatic-cationic peptides described herein are useful to prevent ortreat disease such as atherosclerosis. The combination of peptides andactive agents such as those described above (e.g., antihyperlipedimicagents such as statins) are useful in treating any atherosclerosis, aswell as the signs, symptoms or complications of atherosclerosis.Atherosclerosis (also known as arteriosclerotic vascular disease orASVD) is a condition in which an artery wall thickens as a result of theaccumulation of fatty materials such as cholesterol. Atherosclerosis isa chronic disease that can remain asymptomatic for decades. It is asyndrome affecting arterial blood vessels, a chronic inflammatoryresponse in the walls of arteries, caused largely by the accumulation ofmacrophage white blood cells and promoted by low-density lipoproteins(plasma proteins that carry cholesterol and triglycerides) withoutadequate removal of fats and cholesterol from the macrophages byfunctional high density lipoproteins (HDL). It is commonly referred toas a hardening or furring of the arteries. It is caused by the formationof multiple plaques within the arteries.

The pathobiology of atherosclerotic lesions is complicated butgenerally, stable atherosclerotic plaques, which tend to beasymptomatic, are rich in extracellular matrix and smooth muscle cells,while unstable plaques are rich in macrophages and foam cells and theextracellular matrix separating the lesion from the arterial lumen (alsoknown as the fibrous cap) is usually weak and prone to rupture. Rupturesof the fibrous cap expose thrombogenic material, such as collagen to thecirculation and eventually induce thrombus formation in the lumen. Uponformation, intraluminal thrombi can occlude arteries outright (e.g.,coronary occlusion), but more often they detach, move into thecirculation and can eventually occlude smaller downstream branchescausing thromboembolism (e.g., stroke is often caused by thrombusformation in the carotid arteries). Apart from thromboembolism,chronically expanding atherosclerotic lesions can cause complete closureof the lumen. Chronically expanding lesions are often asymptomatic untillumen stenosis is so severe that blood supply to downstream tissue(s) isinsufficient resulting in ischemia.

These complications of advanced atherosclerosis are chronic, slowlyprogressive and cumulative. In some instances, soft plaques suddenlyrupture, causing the formation of a thrombus that will rapidly slow orstop blood flow, leading to death of the tissues fed by the artery(infarction). Coronary thrombosis of a coronary artery is also a commoncomplication which can lead to myocardial infarction. Blockage of anartery to the brain may result in stroke. In advanced atheroscleroticdisease, claudication from insufficient blood supply to the legs,typically caused by a combination of both stenosis and aneurysmalsegments narrowed with clots, may occur.

Atherosclerosis can affect the entire artery tree, but larger,high-pressure vessels such as the coronary, renal, femoral, cerebral,and carotid arteries are typically at greater risk.

Signs, symptoms and complications of atherosclerosis include, but arenot limited to increased plasma total cholesterol, VLDL-C, LDL-C, freecholesterol, cholesterol ester, triglycerides, phospholipids and thepresence of lesions (e.g., plaques) in arteries, as discussed above. Insome embodiments, increased cholesterol (e.g., total cholesterol, freecholesterol and cholesterol esters) can be seen in one or more ofplasma, aortic tissue and aortic plaques.

Predisposion to atherosclerosis is also a concern. Accordingly, thepresent disclosure relates to methods of administering aromatic-cationicpeptides alone, or in combination with one or more antihyperlipidemicagents (e.g., statins), to prevent atherosclerosis, or the signs,symptoms or complications thereof. In some embodiments a subjectpredisposed to atherosclerosis may exhibit one or more of the followingcharacteristics: advanced age, a family history of heart disease, abiological condition, high blood cholesterol. In some embodiments, thebiological condition comprises high levels of low-density lipoproteincholesterol (LDL-C) in the blood, low levels of high-density lipoproteincholesterol (HDL-C) in the blood, hypertension, insulin resistance,diabetes, excess body weight, obesity, sleep apnea, lifestyle choiceand/or a behavioral habit. In some embodiments, the behavioral habitcomprises smoking and/or alcohol use. In some embodiments, the lifestylechoice comprises an inactive lifestyle and/or a high stress level.

Statin-Related Side Effects

In some embodiments, aromatic-cationic peptides of the presentdisclosure (e.g., D-Arg-2′6′-Dmt-Lys-Phe-NH₂), or a pharmaceuticallyacceptable salt thereof such as acetate salt or trifluoroacetate salt,are administered with one or more hypolipidemic agents (e.g., statins).In some embodiments, the statin and the aromatic-cationic peptide arechemically linked. In some embodiments, the aromatic-cationic peptidesof the present disclosure delay onset, ameliorate, inhibit or eliminatethe side-effects and/or toxicity of hypolipeidemic agent (e.g., statin).In some embodiments, the peptides ameliorate organ damage caused byhypolipidemic agents (e.g., statins). In some embodiments, the peptidesameliorate liver damage, kidney damage, renal toxicity orrhabdomyolysis. In some embodiments, the peptides ameliorate symptomsassociated with the toxic side effects of hypolipidemic agents,including but not limited to muscle weakness, muscle tenderness,malaise, headache, fever, dark urine, nausea, and vomiting.

Hypolipidemic Agent Dosage

In some embodiments, administration of aromatic-cationic peptides of thepresent technology in conjunction with one or more hypolipidemic agents(e.g., statins), permits a higher dose of the hypolipidemic agent to beadministered to a subject than would otherwise be tolerated by thesubject. Also disclosed herein are methods for increasing anantihyperlipidemic agent (such as a statin) dose in a subject in needthereof, or allowing administration of an antihyperlipidemic agent (suchas a statin) to a subject who would normally be contraindicated fore.g., statin treatment (e.g., in a subject who exhibits negative sideeffects related to statin administration at an effective dose).Exemplary negative side effects are described above and in more detailbelow, and include but are not limited to muscle weakness and organdamage.

In some embodiments, by ameliorating the toxic or negative side effectsof the hypolipidemic agent, the dose of hypolipidemic agent may beincreased to a level sufficient to achieve a target blood lipid level.

In some embodiments, the target blood lipid level is a total cholesterollevel. In some embodiments, the target cholesterol level is less thanabout 200 mg/dL. In some embodiments, the target cholesterol level isfrom about 130 to about 200 mg/dL. In some embodiments, the targetcholesterol level is less than about 200, less than about 190, less thanabout 180, less than about 170, less than about 160, less than about150, less than about 140, or less than about 130 mg/dL.

Additionally or alternatively, in some embodiments, the target bloodlipid level is a target LDL level. In some embodiments, the target LDLlevel is less than about 100 mg/dL. In some embodiments, the target LDLlevel is from about 50 to about 100 mg/dL. In some embodiments, thetarget LDL level is less than about 100, less than about 90, less thanabout 80, less than about 70, less than about 60, or less than about 50mg/dL.

Additionally or alternatively, in some embodiments, the target bloodlipid level is a target HDL level. In some embodiments, the target HDLlevel is greater than about 60 mg/dL. In some embodiments, the targetHDL level is from about 30 to about 65 mg/dL. In some embodiments, thetarget HDL level is greater than about 30, greater than about 35,greater than about 40, greater than about 45, greater than about 50,greater than about 55, greater than about 60, or greater than about 65mg/dL.

Additionally or alternatively, in some embodiments, the target bloodlipid level is a target triglyceride level. In some embodiments, thetarget triglyceride level is less than about 200 mg/dL. In someembodiments, the target triglyceride level is from about 140 to about200 mg/dL. In some embodiments, the target triglyceride level is lessthan about 140, less than about 150, less than about 160, less thanabout 170, less than about 180, less than about 190, or less than about200 mg/dL.

By way of example, but not by way of limitation, in some embodiments, asubject with an unsuitable/unhealthy lipid level is administered a firstdosage level of an anithyperlipidemic agent (e.g., a statin) to achievea target lipid level, in combination with a first dosage level of anaromatic-cationic peptide of the present disclosure (e.g.,D-Arg-2′6′-Dmt-Lys-Phe-NH₂) at t=0. In some embodiments, the subject isadministered the anithyperlipidemic agent, or is administered ananithyperlipidemic agent and a peptide at the first dosage level for 1day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month,2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, 4years, 5 years or 10 years. In some embodiments, the subject isadministered the anithyperlipidemic agent, or is administered ananithyperlipidemic agent and a peptide at the first dosage level onceper day, twice per day, every other day, once every third day, fourthday, fifth day, once per week, or once every other week. In someembodiments, the antihyperlipidemic agent and the aromatic-cationicpeptide are chemically linked.

At a later time (t=1) (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 1week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year or2 years) after taking the anithyperlipidemic agent, or theanithyperlipidemic agent and the peptide at the first dosage level, thesubject's lipid levels and any negative or toxic side effectscharacteristic of the anithyperlipidemic agent are evaluated. In someembodiments, due to the positive effects of the peptpide, the subjectexhibits no negative or toxic side effects of the anithyperlipidemicagent. In some embodiments, the dosage level of the antihyperlipidemicagent is increased to a second dosage level to more quickly oreffectively achieve an acceptable (e.g., target) lipid level. In someembodiments, the peptide dosage level remains constant, e.g., is equalto the first dosage level. In some embodiments, the peptide dosage levelis increased, e.g., is greater than the first dosage level. In someembodiments, the peptide dosage level is decreased, e.g., is less thanthe first dosage level. In some embodiment, no additional peptide isadministered with the second dosage level of the antihyperlipidemicagent. In some embodiments, peptide is administered as often as theantihyperlipidemic agent. In some embodiments, the peptide isadministered more or less frequently than the antihyperlipidemic agent.In some embodiments, the antihyperlipidemic agent and thearomatic-cationic peptide are chemically linked.

In some embodiments, the subject's lipid levels and any negative ortoxic side effects characteristic of the anithyperlipidemic agent areevaluated at t=2, t=3, etc. In some embodiments, the dosage level of theantihyperlipidemic agent is increased to a third, fourth, fifth, etc.dosage level to more quickly or effectively achieve a target lipidlevel. In some embodiments, at t=2, 3, etc. the peptide dosage level maydecreased, increased, remain the same (e.g., first dosage level) or beomitted from one or more administrations.

Coenzyme Q10 Levels

The statins (simvastatin, lovastatin, pravastatin, fluvastatin and thelike) are hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductaseinhibitors. By inhibiting this enzyme, statins reduce the synthesis ofmevalonate, an intermediary in the cholesterol synthesis pathway. Thesame biosynthetic pathway is shared by coenzyme Q10; mevalonate is alsoa precursor of coenzyme Q10. Thus, both cholesterol and coenzyme Q10biosynthesis decrease with statin treatment.

Some of the side effects of statins include mitochondrial dysfunction,decreased coenzyme Q10 levels, a variety of myopathies (ranging frommild myalgia to fatal rhabdomyolysis), diabetes, kidney failure andmemory loss. Additional side effects include fever, dark colored urine,swelling, weight gain, changes in urination frequency, dry mouth,drowsiness, nausea, diarrhea, jaundice, loss of appetite, insomnia, andheadache.

Coenzyme Q10 (CoQ10) is a naturally occurring, fat-soluble quinone thatis localized in hydrophobic portions of cellular membranes.Approximately half of the body's CoQ10 is obtained through dietary fatingestion, whereas the remainder results from endogenous synthesis.Coenzyme Q10 participates in electron transport during oxidativephosphorylation in mitochondria, protects against oxidative stressproduced by free radicals, and regenerates active forms of theantioxidants ascorbic acid and tocopherol (vitamin E). Given the role ofCoQ10 in mitochondrial energy production and the importance ofmitochondria in muscle function, it is likely that statin-induced CoQ10deficiency plays a role in statin-associated mitochondrial dysfunctionand myopathies (e.g., rhabdomyolysis). Without wishing to be bound bytheory, it is also possible that CoQ10 plays a role in additionalstatin-induced side effects, such as but not limited to memory loss,kidney failure and diabetes.

As shown in Example 3 and FIG. 5, aromatic-cationic peptides of thepresent disclosure increase CoQ10 levels in fibroblast cells.Accordingly, in some embodiments, aromatic-cationic peptides of thepresent disclosure are administered with one or more statins toalleviate or prevent the myopathic side effects of statinadministration. The peptide may be administered before, simultaneouslywith, or after statin administration. The reason for statinadministration is not intended to limit peptide administration. That is,the subject may be suffering from, or at risk for, any number ofdisease, conditions or illnesses for which one or more statins areindicated.

By way of example, but not by way of limitation, exemplary diseases,conditions, risk factors, characteristics, or reasons for administeringa statin include one or more of the following: advanced age, smoking,hypertension, low HDL-C, a family history of early coronary heartdisease, an increased risk of myocardial infarction and stroke insubjects with type 2 diabetes without coronary heart disease, but withother or multiple risk factors (e.g., retinopathy, albuminuria, smoking,or hypertension), to reduce the risk of non-fatal MI, fatal andnon-fatal stroke, revascularization procedures, hospitalization forcoronary heart failure, or angina in patients with coronary heartdisease, to reduce elevated total cholesterol, LDL-C, ApoB, andtriglyceride levels and increase HDL-C in adult patients with primaryhyperlipidemia (heterozygous familial and nonfamilal) and mixeddyslipidemia, to reduce elevated triglycerides in patients withhypertriglyceridemia and primary dysbetalipoproteinemia, to reduce totalcholesterol and LDL-C in patients with homozygous familialhypercholesterolemia (HoFH), to reduce elevated total cholesterol,LDL-C, and ApoB levels in boys and postmenarchal girls between the agesof 10-17, with heterozygous familial hypercholesterolemia after failingan adequate trial of diet therapy, to treat patients with primaryhyperlipidemia and mixed dyslipidemia as an adjunct to diet to reducelevels of total cholesterol, LDL-C, ApoB, nonHDL-C, and triglyceridelevels and to increase levels of HDL-C, to treat patients with:hypertriglyceridemia as an adjunct to diet, primarydysbetalipoproteinemia (Type II hyperlipoproteinemia) as an adjunct todiet, and homozygous familial hypercholesterolemia (HoFH), to slow theprogression of atherosclerosis in patients as part of a treatmentstrategy to lower total cholesterol and LDL-C as an adjunct to diet, totreat patients 10 to 17 years old with heterozygous familialhypercholesterolemia (HeFH) to reduce elevated total cholesterol, LDL-C,and ApoB after failing an adequate trial of diet therapy, to reduce therisk of myocardial infarction, stroke, and arterial revascularizationprocedures in patients without evident coronary heart disease, but withmultiple risk factors (e.g., hypertension, low HDL-C, smoking, or afamily history of premature coronary heart disease), to reduceinflammation, promote locomotion, and promote tissue sparing in spinalcord injury, and/or to reduce or eliminate infection of HCV in apatient.

In addition, in some embodiments, the administration of one or morearomatic-cationic peptides of the present disclosure in combination withone or more antihyperlipidemic agents (e.g., statins) will allow thesubject to receive a higher dose of one or more antihyperlipidemicagents to alleviate a disease, conditions, or a sign, symptom orcharacteristic of a disease or condition. By way of example but not byway of limitation, the label on the statin CRESTOR® emphasizes the risks(e.g., myopathy, rhabdomyolysis and various forms of kidney failure) atthe highest approved does of 40 mg, and recommends administration oflower doses. By administering an aromatic-cationic peptide with theantihyperlipidemic agent, the detrimental side effects seen with higherdosages may delayed, ameliorated or eliminated, thereby allowing foradministration of the higher therapeutic antihyperlipidemic (e.g.,statin) dose. In some embodiments, the anithyperlipidemic agent and thearomatic-cationic peptide are chemically linked.

General

The disclosure also provides for both prophylactic and therapeuticmethods of treating a subject having or at risk of (or susceptible to)atherosclerosis and related complications. Accordingly, the presentmethods provide for the prevention and/or treatment of atherosclerosisin a subject by administering an effective amount of anaromatic-cationic peptide and one or more active agents, such as anantihyperlipidemic drug (e.g., a statin) to a subject in need thereof.In some embodiments, the anithyperlipidemic agent and thearomatic-cationic peptide are chemically linked.

In various embodiments, suitable in vitro or in vivo assays areperformed to determine the effect of a specific combination ofaromatic-cationic peptides and one or more active agents and whether itsadministration is indicated for treatment. In various embodiments,assays can be performed with representative animal models to determineif a given aromatic-cationic peptide and cardiovascular agent treatmentregime exerts the desired effect in preventing or treatingatherosclerosis. Compounds for use in therapy can be tested in suitableanimal model systems including, but not limited to rats, mice, chicken,pigs, cows, monkeys, rabbits, and the like, prior to testing in humansubjects. Any of the animal model systems known in the art can be usedprior to administration to human subjects.

In therapeutic applications, compositions or medicaments areadministered to a subject suspected of, or already suffering from such adisease in an amount sufficient to cure, or at least partially arrest,the symptoms of the disease, including its complications andintermediate pathological phenotypes in development of the disease. Assuch, the invention provides methods of treating an individual afflictedwith atherosclerosis.

Modes of Administration, Formulations and Effective Dosages

Formulations

Any method known to those in the art for contacting a cell, organ ortissue with a peptide and active agent may be employed. Suitable methodsinclude in vitro, ex vivo, or in vivo methods. In vivo methods typicallyinclude the administration of an aromatic-cationic peptide and activeagent, such as those described above, to a mammal, suitably a human.When used in vivo for therapy, the aromatic-cationic peptides and activeagents may be administered to the subject in effective amounts (i.e.,amounts that have desired therapeutic effect). The dose and dosageregimen will depend upon the degree of the injury in the subject, thecharacteristics of the particular aromatic-cationic peptide and/oractive agent used, e.g., its therapeutic index, the subject, and thesubject's history.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of a peptide and one or more additional active agentsuseful in the methods may be administered to a mammal in need thereof byany of a number of well-known methods for administering pharmaceuticalcompounds. The peptide may be administered systemically or locally.

The compound may be formulated as a pharmaceutically acceptable salt.The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (e.g., salts having acceptable mammalian safety for agiven dosage regime). However, it is understood that the salts are notrequired to be pharmaceutically acceptable salts, such as salts ofintermediate compounds that are not intended for administration to apatient. Pharmaceutically acceptable salts can be derived frompharmaceutically acceptable inorganic or organic bases and frompharmaceutically acceptable inorganic or organic acids. In addition,when a peptide contains both a basic moiety, such as an amine, pyridineor imidazole, and an acidic moiety such as a carboxylic acid ortetrazole, zwitterions may be formed and are included within the term“salt” as used herein. Salts derived from pharmaceutically acceptableinorganic bases include ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic, manganous, potassium, sodium, and zincsalts, and the like. Salts derived from pharmaceutically acceptableorganic bases include salts of primary, secondary and tertiary amines,including substituted amines, cyclic amines, naturally-occurring aminesand the like, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically acceptable inorganicacids include salts of boric, carbonic, hydrohalic (hydrobromic,hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamicand sulfuric acids. Salts derived from pharmaceutically acceptableorganic acids include salts of aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (e.g., aspartic and glutamicacids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic,diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like. In some embodiments, the pharmaceutically acceptable saltis acetate or trifluoroacetate salt.

The compounds described herein can be incorporated into pharmaceuticalcompositions for administration, singly or in combination, to a subjectfor the treatment or prevention of a disorder described herein. Suchcompositions typically include the active agent (e.g., peptide and oneor more active agents, e.g., a statin) and a pharmaceutically acceptablecarrier. As used herein the term “pharmaceutically acceptable carrier”includes saline, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. For convenience of thepatient or treating physician, the dosing formulation can be provided ina kit containing all necessary equipment (e.g., vials of drug, vials ofdiluent, syringes and needles) for a treatment course.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The pharmaceutical compositions can include a carrier, which can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thiomerasol, and the like. Glutathione and other antioxidants can beincluded to prevent oxidation. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompounds can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include, but are not limited to those describedin U.S. Pat. No. 6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. In one embodiment, transdermal administration may be performedby iontophoresis.

A therapeutic agent can be formulated in a carrier system. The carriercan be a colloidal system. The colloidal system can be a liposome, aphospholipid bilayer vehicle. In one embodiment, the therapeutic peptideis encapsulated in a liposome while maintaining peptide integrity. Asone skilled in the art would appreciate, there are a variety of methodsto prepare liposomes. (See Lichtenberg et al., Methods Biochem. Anal.,33:337-462 (1988); Anselem et al., Liposome Technology, CRC Press(1993)). Liposomal formulations can delay clearance and increasecellular uptake (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)).An active agent can also be loaded into a particle prepared frompharmaceutically acceptable ingredients including, but not limited to,soluble, insoluble, permeable, impermeable, biodegradable orgastroretentive polymers or liposomes. Such particles include, but arenot limited to, nanoparticles, biodegradable nanoparticles,microparticles, biodegradable microparticles, nanospheres, biodegradablenanospheres, microspheres, biodegradable microspheres, capsules,emulsions, liposomes, micelles and viral vector systems.

The carrier can also be a polymer, e.g., a biodegradable, biocompatiblepolymer matrix. In one embodiment, the therapeutic peptide can beembedded in the polymer matrix, while maintaining protein integrity. Thepolymer may be natural, such as polypeptides, proteins orpolysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of, e.g., collagen, fibronectin, elastin,cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,and combinations thereof. In one embodiment, the polymer is poly-lacticacid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matricescan be prepared and isolated in a variety of forms and sizes, includingmicrospheres and nanospheres. Polymer formulations can lead to prolongedduration of therapeutic effect. (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). A polymer formulation for human growth hormone(hGH) has been used in clinical trials. (See Kozarich and Rich, ChemicalBiology, 2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale et al.), PCT publication WO96/40073 (Zale et al.), and PCT publication WO 00/38651 (Shah et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylacetic acid. Such formulations can be preparedusing known techniques. The materials can also be obtained commercially,e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions (including liposomes targeted to specific cells withmonoclonal antibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described in,but not limited to U.S. Pat. No. 4,522,811.

The therapeutic compounds can also be formulated to enhanceintracellular delivery. For example, liposomal delivery systems areknown in the art, see, e.g., Chonn and Cullis, “Recent Advances inLiposome Drug Delivery Systems,” Current Opinion in Biotechnology6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: SelectingManufacture and Development Processes,” Immunomethods, 4(3):201-9(1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery:Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995).Mizguchi et al., Cancer Lett., 100:63-69 (1996), describes the use offusogenic liposomes to deliver a protein to cells both in vivo and invitro.

Formulations Linking Peptides and Additional Active Agents—CombinationTherapy

In some embodiments, at least one additional active agent, e.g., anantihyperlipidemic agent (e.g., statin), and at least one aromaticcationic peptide as described above (e.g., D-Arg-2′6′-Dmt-Lys-Phe-NH₂ ora pharmaceutically acceptable salt thereof), are associated to form acomplex. The antihyperlipidemic agent and aromatic-cationic peptide canassociate by any method known to those in the art. The followingexamples of peptide/active agent linkages are provided by way ofillustration only, and are not inteneded to be limiting. In general,additional active agents can be linked to an aromatic-cationic peptideof the present disclosure by any suitable technique, with appropriateconsideration of the need for pharmokinetic stability and reducedoverall toxicity to the subject. A therapeutic agent can be coupled toan aromatic-cationic peptide either directly or indirectly (e.g., via alinker group).

Suitable types of associations include chemical bonds and physicalbonds. Chemical bonds include, for example, covalent bonds andcoordinate bonds. Physical bonds include, for instance, hydrogen bonds,dipolar interactions, van der Waal forces, electrostatic interactions,hydrophobic interactions and aromatic stacking. In some embodiments,bonds between the compounds are rapidly degraded or dissolved; in someembodiments, bonds are cleaved by drug metabolizing or excretatorychemistry and/or enzymes.

For a chemical bond or physical bond, a functional group on the moleculetypically associates with a functional group on the aromatic cationicpeptide. For example, hypolipidemic agents such as statins often containa carboxyl functional group, as well as hydroxyl functional groups. Thefree amine group of an aromatic cationic peptide may be crosslinkeddirectly to the carboxl group of a statin using1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC orEDAC) or dicyclohexylcarbodiimide (DCC). Cross-linking agents can, forexample, be obtained from Pierce Biotechnology, Inc., Rockford, Ill. ThePierce Biotechnology, Inc. website can provide assistance.

In some embodiments, a direct reaction between an additional activeagent (e.g., an antihyperlipidemic agent) and an aromatic-cationicpeptide (e.g., D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceuticallyacceptable salt thereof), is formed when each possesses a functionalgroup capable of reacting with the other. For example, a nucleophilicgroup, such as an amino or sulfhydryl group, can be capable of reactingwith a carbonyl-containing group, such as an anhydride or an acidhalide, or with an allyl group containing a good leaving group (e.g., ahalide). Additionally or alternatively, a suitable chemical linker groupcan be used. A linker group can function as a spacer to distance thepeptide and the additional active agent in order to avoid interferencewith, for example, binding capabilities. A linker group can also serveto increase the chemical reactivity of a substituent, and thus increasethe coupling efficiency.

In exemplary embodiments, suitable linkage chemistries includemaleimidyl linkers and alkyl halide linkers (which react with asulfhydryl on the antibody moiety) and succinimidyl linkers (which reactwith a primary amine on the antibody moiety). Several primary amine andsulfhydryl groups are present on immunoglobulins, and additional groupscan be designed into recombinant immunoglobulin molecules. It will beevident to those skilled in the art that a variety of bifunctional orpolyfunctional reagents, both homo- and hetero-functional (such as thosedescribed in the catalogue of the Pierce Chemical Co., Rockford, Ill.),can be employed as a linker group. Coupling can be affected, e.g.,through amino groups, carboxyl groups, sulfhydryl groups or oxidizedcarbohydrate residues (see, e.g., U.S. Pat. No. 4,671,958).

As an additional or alternative coupling method, an additional activeagent can be coupled to the aromatic-cationic peptides disclosed herein,e.g., through an oxidized carbohydrate group at a glycosylation site,for example, as described in U.S. Pat. Nos. 5,057,313 and 5,156,840. Yetanother alternative method of coupling an aromatic cationic peptide toan additional active agent is by the use of a non-covalent binding pair,such as streptavidin/biotin, or avidin/biotin. In these embodiments, onemember of the pair is covalently coupled to the aromatic-cationicpeptide, and the other member of the binding pair is covalently coupledto the additional active agent.

In some embodiments, an additional active agent may be more potent whenfree from the aromatic-cationic peptide, and it may be desirable to usea linker group which is cleavable during or upon internalization into acell, or which is gradually cleavable over time in the extracellularenvironment. A number of different cleavable linker groups have beendescribed. Examples of the intracellular release of active agents fromthese linker groups include, e.g., but are not limited to, cleavage byreduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014), byhydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No.4,638,045), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No.4,671,958), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789).

In some embodiments, an aromatic-cationic peptide as disclosed herein iscoupled to more than one active agent. For example, in some embodiments,aromatic-cationic peptide is coupled to a mixture of at least twoadditional active agents. That is, more than one type of active agentcan be coupled to one aromatic-cationic peptide. For instance, atherapeutic moiety, such as a an antihyperlipidemic agents such as astatin, polynucleotide, antibody or antisense sequence, can beconjugated to an aromatic-cationic peptide to increase the effectivenessof the therapy, as well as lowering the required dosage necessary toobtain the desired therapeutic effect. Regardless of the particularembodiment, formulations with more than one moiety can be prepared in avariety of ways. For example, more than one moiety can be coupleddirectly to an aromatic-cationic peptide, or linkers that providemultiple sites for attachment (e.g., dendrimers) can be used.Alternatively, a carrier with the capacity to hold more than one activeagent can be used.

As explained above, an aromatic-cationic peptide can be linked toadditional active agents in a variety of ways, including covalentbonding either directly or via a linker group, and non-covalentassociations. For example, in some embodiments, the aromatic-cationicpeptide and additional active agents can be combined with encapsulationcarriers. In some embodiments, this is especially useful to allow thetherapeutic compositions to gradually release the aromatic-cationicpeptide and additional active agent over time while concentrating it inthe vicinity of the target cells.

In some embodiments, linkers that that are cleaved within a cell mayalso be used. For example, heterocyclic “self-immolating” linkermoieties can be used to link aromatic cationic peptides of the presentinvention to additional active agents such as antihyperlipidemic drugs,such as statins (see, for example U.S. Pat. No. 7,989,434 and U.S. Pat.No. 8,039,273, incorporated herein by reference).

In some embodiments, the linker moiety comprises a heterocyclic“self-immolating moiety” bound to the aromatic-cationic peptide (e.g.,D-Arg, 2′6′-Dmt-Lys-Phe-NH₂) and an additional active agent (e.g., anantihyperlipidemic agent such as a statin) and incorporates an amidegroup or beta-glucuronide group that, upon hydrolysis by anintracellular protease or beta-glucuronidase, initiates a reaction thatultimately cleaves the self-immolative moiety from the aromatic cationicpeptide such that the additional active agent (e.g., statin) is releasedfrom the peptide in an active form.

Exemplary self immolating moieties include those of Formulas I, II, andIII, presented in FIG. 12. In FIG. 12, the wavy lines indicate thecovalent attachment sites to the aromatic cationic peptide and thestatin, wherein:

-   U is O, S or NR⁶;-   Q is CR⁴ or N;-   V¹, V² and V³ are independently CR⁴ or V¹ provided that for formula    II and III at least one of Q, V¹ and V² is N;-   T is NH, NR⁶, O or S pending from said drug moiety;-   R¹, R², R³ and R⁴ are independently selected from H, F, Cl, Br, I,    OH, —N(R⁵)₂, —N(R⁵)₃, C₁-C₈ alkylhalide, carboxylate, sulfate,    sulfamate, sulfonate, —SO₂R⁵, —S(═O)R⁵, —SR⁵, —SO₂N(R⁵)₂, —C(═O)R⁵,    —CO₂R⁵, —C(═O)N(R⁵)₂, —CN, —N₃, —NO₂, C₁-C₈ alkoxy, C₁-C₈    halosubstituted alkyl, polyethyleneoxy, phosphonate, phosphate,    C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₂-C₈ alkenyl, C₂-C₈    substituted alkenyl, C₂-C₈ alkynyl, C₂-C₈ substituted alkynyl,    C₆-C₂₀ aryl, C₆-C₂₀ substituted aryl, C₁-C₂₀ heterocycle, and C₁-C₂₀    substituted heterocycle; or when taken together, R² and R³ form a    carbonyl (═O), or spiro carbocyclic ring of 3 to 7 carbon atoms; and-   R⁵ and R⁶ are independently selected from H, C₁-C₈ alkyl, C₁-C₈    substituted alkyl, C₂-C₈ alkenyl, C₂-C₈ substituted alkenyl, C₂-C₈    alkynyl, C₂-C₈ substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substituted    aryl, C₁-C₂₀ heterocycle, and C₁-C₂₀ substituted heterocycle;-   where C₁-C₈ substituted alkyl, C₂-C₈ substituted alkenyl, C₂-C₈    substituted alkynyl, C₆-C₂₀ substituted aryl, and C₂-C₂₀ substituted    heterocycle are independently substituted with one or more    substituents selected from F, Cl, Br, I, OH, —N(R⁵)₂, —N(R⁵)₃ ⁺,    C₁-C₈ alkylhalide, carboxylate, sulfate, sulfamate, sulfonate,    C₁-C₈alkylsulfonate, C₁-C₈ alkylamino, 4-dialkylaminopyridinium,    C₁-C₈ alkylhydroxyl, C₁-C₈ alkylthiol, —SO₂R⁵, —S(═O)R⁵, —SR^(S),    —SO₂N(R⁵)₂, C(═O)R⁵, —CO₂R⁵, —C(═O)N(R⁵)₂, —CN, —N₃, —NO₂, C₁-C₈    alkoxy, C₁-C₈ trifluoroalkyl, C₁-C₈ alkyl, C₃-C₁₂ carbocycle, C₆-C₂₀    aryl, C₂-C₂₀ heterocycle, polyethyleneoxy, phosphonate, and    phosphate.

The linker moiety may further include a cleavable peptide sequenceadjacent to the self-immolative moiety that is a substrate for anintracellular enzyme, for example a cathepsin such as cathepsin B, thatcleaves the cleavable peptide at the amide bond shared with theself-immolative moiety (e.g. Phe-Lys, Ala-Phe, or Val-Cit). In someembodiments, the amino acid residue chain length of the cleavablepeptide sequence ranges from that of a single amino acid to about eightamino acid residues. The following are exemplary enzymatically-cleavablepeptide sequences: Gly-Gly, Phe-Lys, Val-Lys, Phe-Phe-Lys,D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit,Trp-Cit, Phe-Ala, Ala-Phe, Gly-Gly-Gly, Gly-Ala-Phe, Gly-Val-Cit,Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-N 9-tosyl-Arg, and Phe-N9-Nitro-Arg, in either orientation. Numerous specific cleavable peptidesequences suitable for use in the present formulations can be designedand optimized in their selectivity for enzymatic cleavage by aparticular intracellular enzyme, e.g. liver cell enzymes.

A spacer unit may be linked to the aromatic cationic peptide via anamide, amine or thioether bond. The additional active agent (e.g., anantihyperlipidemic agent such as a statin) may be connected to theself-immolative moiety of the linker via a chemically reactivefunctional group pending from the additional active agent, such as aprimary or secondary amine, hydroxyl, or carboxyl group.

Additionally or alternatively, the self-immolative linkers describedherein can be used to link aromatic-cationic peptides to antibodies,nucleic acids or other biologically active molecules (e.g.,antihyperlipidemic agents). For example, an aromatic cationic peptidewith or without a spacer may be attached to a linker that includes acleavable peptide sequence or beta-glucuronide group and aself-immolative linker attached to an antibody, e.g., an antibody thatreduces LDL-C, such as an antibody against a PCSK9 inhibitor and/or ananti-ApoB agent, such as an antisense RNA.

Exemplary schematics of illustrative embodiments of such formulationsare shown in FIG. 13.

In some embodiments, once the statin-peptide complex enters the cell orblood stream, the linker is cleaved releasing the peptide from theantihyperlipidemic agent (e.g., a statin). The formulations are notintended to be limited by linkers or cleavage means. For example, insome embodiments, linkers are cleaved in the body (e.g., in the bloodstream, interstitial tissue, gastrointestinal tract, etc.), releasingthe peptide from the second active agent (e.g., an antihyperlipidemicdrug such as a statin) via enzymes (e.g., esterases) or other chemicalreactions.

Statins—

As noted above, in one aspect, the present disclosure providescombination therapies for the treatment of atherosclerosis comprisingadministering an effective amount of peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂or a pharmaceutically acceptable salt, such as acetate ortrifluoroacetate salt in combination with one or more statins. In someembodiments the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ is chemically linkedto the one or more statins. In some embodiments, the peptide is linkedto the statin using a labile bond such that hydrolysis in vivo releasesthe two pharmaceutically active agents. A schematic diagram illustratingexemplary embodiments is shown in FIG. 7. In some embodiments, thelinkage comprises an ester, a carbonate, a carbamate or other labilelinkage. FIG. 8 shows illustrative embodiments where when X═O, acarbonate linked pro-drug is formed. One skilled in the art wouldunderstand that where X═NH, a carbamate linked pro-drug is formed.Potential reactive sites on the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ andstatins are shown in FIG. 9.

By way of example but not by way of limitation, FIG. 10 illustrates howD-Arg-2′6′-Dmt-Lys-Phe-NH₂ could be linked to either CRESTOR® orLIPITOR® using a carbonate linkage. By way of example but not by way oflimitation, FIG. 11 illustrates how D-Arg-2′6′-Dmt-Lys-Phe-NH₂ could belinked to CRESTOR® using a carbamate linkage.

PCSK9—

In some embodiments, the present disclosure provides combinationtherapies comprising administering an effective amount of peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable salt, suchas acetate or trifluoroacetate salt in combination with one or moretherapies targeting PCSK9. In some embodiments, theD-Arg-2′6′-Dmt-Lys-Phe-NH₂ peptide is conjugated to an anti-PCSK9antibody to form a peptide-antibody conjugate. A variety of bifunctionalprotein coupling agents may be used. By way of example, but not by wayof limitation, in some embodiments, the peptide-antibody conjugates areprepared using one or more of N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), or bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

As noted above, in some embodiments, the peptide-antibody conjugate isgenerated using a cleavable linker to facilitate release of the peptidein vivo. In some embodiments, the cleavable linker is an acid-labilelinker, peptidase-sensitive linker, photolabile linker, a dimethyllinker, or a disulfide-containing linker. In some embodiments, thelinker is a labile linkage that is hydrolyzed in vivo to release theantibody and peptide. In some embodiments, the labile linkage comprisesan ester linkage, a carbonate linkage, or a carbamate linkage.

Anti-Apo-B—

In some embodiments, the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ ischemically linked to an anti-Apo-B agent (e.g., an anti-Apo-B RNA, e.g.,an antisense RNA) using a labile linkage to form a pro-drug that uponhydrolysis in vivo releases the peptide and the anti-Apo-B agent asactive agents. In some embodiments, the labile linkage comprises anester linkage, a carbonate linkage, or a carbamate linkage. By way ofillustration but not by way of limitation, FIG. 9 shows potentialreactive sites on the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ at which ananti-Apo-B agent could be linked.

Additional Formulations—Combination Therapy

In some embodiments, the antihyperlipidemic agent and an aromaticcationic peptide of the present disclosure may be administered in theform of a pharmaceutical composition comprising at least one of thecompounds disclosed herein together with a pharmaceutically acceptablecarrier or diluent. Thus, in some embodiments, the compounds disclosedherein can be administered either individually or together in anyconventional oral, parenteral or transdermal dosage form. In someembodiments, the antihyperlipidemic agent may be co-formulated in afixed-dose combination with the aromatic cationic peptide. In someembodiments, the antihyperlipidemic agent and the aromatic cationicpeptide are formulated in a capsule or pill for oral dosing in which thecompounds are physically separated. In such a formulation, one or bothof the antihyperlipidemic agent and the aromatic cationic peptide are ina solid, liquid, powder, or gel form. In some embodiments, theantihyperlipidemic agent and the aromatic cationic peptide are in afixed-dose combination in which the two compounds are mixed together,for example in in a solid, liquid, powder, or gel form.

Dosage

Dosage, toxicity and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

Typically, an effective amount of the aromatic-cationic peptides and/orcardiovascular agents, sufficient for achieving a therapeutic orprophylactic effect, range from about 0.000001 mg per kilogram bodyweight per day to about 10,000 mg per kilogram body weight per day.Preferably, the dosage ranges are from about 0.0001 mg per kilogram bodyweight per day to about 100 mg per kilogram body weight per day. Forexample dosages can be 1 mg/kg body weight or 10 mg/kg body weight everyday, every two days or every three days or within the range of 1-10mg/kg every week, every two weeks or every three weeks. In oneembodiment, a single dosage of peptide ranges from 0.1-10,000 microgramsper kg body weight. In one embodiment, aromatic-cationic peptideconcentrations in a carrier range from 0.2 to 2000 micrograms perdelivered milliliter.

In some embodiments, a therapeutically effective amount of anaromatic-cationic peptide may be defined as a concentration of peptideat the target tissue of 10⁻¹¹ to 10⁻⁶ molar, e.g., approximately 10⁻⁷molar. This concentration may be delivered by systemic doses of 0.01 to100 mg/kg or equivalent dose by body surface area. The schedule of doseswould be optimized to maintain the therapeutic concentration at thetarget tissue, most preferably by single daily or weekly administration,but also including continuous administration (e.g., parenteral infusionor transdermal application).

In some embodiments, the dosage of the aromatic-cationic peptide isprovided at a “low,” “mid,” or “high” dose level. In one embodiment, thelow dose is provided from about 0.0001 to about 0.5 mg/kg/h, suitablyfrom about 0.001 to about 0.1 mg/kg/h. In one embodiment, the mid-doseis provided from about 0.01 to about 1.0 mg/kg/h, suitably from about0.01 to about 0.5 mg/kg/h. In one embodiment, the high dose is providedfrom about 0.5 to about 10 mg/kg/h, suitably from about 0.5 to about 2mg/kg/h. In an illustrative embodiment, the dose of cardiovascular agentis from about 1 to 100 mg/kg, suitably about 25 mg/kg.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The mammal treated in accordance present methods can be any mammal,including, for example, farm animals, such as sheep, pigs, cows, andhorses; pet animals, such as dogs and cats; laboratory animals, such asrats, mice and rabbits. In some embodiments, the mammal is a human.

EXAMPLES

The present invention is further illustrated by the following example,which should not be construed as limiting in any way.

Example 1 Effects of Aromatic-Cationic Peptides in Protecting AgainstAtherosclerosis in a Mouse Model

The effects of aromatic-cationic peptides in protecting againstatherosclerosis in a mouse model were investigated.

Apoprotein E deficient mice (Jackson Laboratories, 600 Main Street, BarHarbor, Me.) were used in this study. The mice were male, 7-8 weeks ofage, and between 18-20 g in weight. An initial total cholesterolmeasurement was made on 30 mice, and the mice were grouped into twogroups of 15 to match the total cholesterol measurements. Both groupswere fed a “western diet” (40 kcal % butterfat, 0.15% [wt/wt]cholesterol, Harlan Teklad diet TD-88137). Starting at t=0, the controlgroup of 15 mice received vehicle only (phosphate buffered saline at pH7.4), while the test group of 15 mice received aromatic-cationic peptidereconstituted in phosphate buffered saline. Body weights of the micewere recorded weekly, and mortality checks were performed daily.

The aromatic-cationic peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ (sterilelyophilized powder, reconstituted in phosphate buffered saline) wastested. For the 12-week study, test mice received a single, daily doseof the peptide, subcutaneously at 1 mg/kg. Control mice received asingle daily dose of vehicle.

Experimental Protocol/Data Collection.

Blood was collected every four weeks (orbital plexis) under isofluraneanesthesia (3%) and blood lipids were determined. Plasma lipid analysiswas conducted for both groups at t=0, 4, 6, 8, and 12 weeks. Plasmalipid analysis using an autoanalyzer included total cholesterol (TC),triglycerides (Trigs), phospholipids (PL), free cholesterol (FC), andcholesterol ester (CE, by calculation). Gel electrophoresis was used tomeasure levels of high-density lipoprotein cholesterol (HDL-C),low-density lipoprotein cholesterol (LDL-C), and very low-densitylipoprotein cholesterol (VLDL-C).

Histopathology/Histomorphometery:

Following the 12 week treatment, mice were euthanized within 48 hoursafter the last dose by CO₂ asphyxiation and the vascular tree wasperfused with 5 mL of phosphate buffered saline (pH 7.4). The aorta andaortic sinus were removed for examination. Thoracic aorta were isolated,trimmed of fat, and fixed in formalin for 48-72 hours before analysis.For en face analysis, aortas were laid out and pinned on black matrixfor photography, and stained with Sudan IV. Vessels were imaged forsurface involvement using a Nikon computerized image analysis system andthe percent of the aortic surface area covered by lipid was calculated.Two determinations were done for each image (Quan 1 and Quan 2), and theaverage was computed. The data were then computed by group andstatistically analyzed. Following staining and morphometric analysis,total lipids were extracted from the aortas using the Bligh-Dyer method.WAKO Diagnostics kits (WAKO Diagnostics, Inc., 1600 Bellwood RoadRichmond, Va. 23237-1326) were then used to evaluate total cholesterol,free cholesterol, and cholesterol ester. Cholesterol leves werequantitate nad expressed relative to protein levels. Values areexpressed as μg lipid per mg protein (see e.g., FIG. 1).

For the aortic sinus, the heart and approximately 5 mm of the ascendingaorta was cut from the remainder of the aorta. The apex of the heart wasremoved and remaining heart with the attached aortic segment was fixedand sectioned (in OCT medium and frozen in a dry ice—2 methylbutanebath). Serial 10 μm thick cryosections were made beginning with theascending aorta and proceeding through the entire aortic sinus until theventricular chamber was reached. The sections were stained with Oil RedO or Sudan IV and counter stained with Harris hematoxylin. Alternatesections were stained with hematoxylin and eosin. The sinus was imagedat 5 step levels in the region of interest, i.e., the aortic root, for atotal distance of 300 μm and the lipid-staining areas and measured(total cross sectional area) using a Nikon computerized image analysissystem. The data were then computed by group and statistically analyzed.

Results are shown in the tables below and in FIGS. 1-4. Table 7 showsthe en face analysis of atherosclerotic lesions for each mouse in thestudy. Column 1: (sample ID #) represents each mouse, 1-15 are controlanimals (received vehicle alone), and 16-20 are test animals (receivedaromatic-cationic peptide); column 2: (Quan 1) shows a firstdetermination of % of the aortic surface covered by lipid for each mousein the study; column 3 (Quan 2) shows a second determination of % of theaortic surface covered by lipid for each mouse in the study; column 4:shows the average % of the surface of the aorta showing lesions for eachmouse in the study; column 5 shows the average % of the surface of theaortas of control or test mice showing lesions; column 5 shows thestandard error of the mean for each group (control or test animals). Asshown in Table 7, treatment with the aromatic-cationic peptide reducesatherosclerotic lesions in the aorta. Aromatic-cationic peptides of thepresent disclosure are therefore useful in treating atherosclerosis andrelated signs, symptoms and complications of atherosclerosis.

TABLE 7 En Face Analysis of Atherosclerotic Lesions Sample Ave. % of ID#Quan 1 Quan 2 Lesion Ave SEM 1 5.810 6.517 6.164 7.189 0.990 2 4.5015.089 4.795 3 2.637 3.567 3.102 4 3.416 3.719 3.568 5 18.228 17.11517.672 6 2.951 3.919 3.435 7 11.164 10.464 10.814 8 4.492 4.538 4.515 96.002 6.080 6.041 10 6.303 5.610 5.957 11 10.293 12.311 11.302 12 8.0447.019 7.532 13 9.162 8.497 8.830 14 8.610 7.629 8.120 15 6.000 5.9815.991 16 2.562 2.667 2.615 5.328 1.027 17 0.914 0.870 0.892 18 8.7699.284 9.027 19 2.832 3.205 3.019 20 7.137 6.881 7.009 21 5.198 4.4684.833 22 2.128 1.863 1.996 23 1.388 1.021 1.205 24 10.000 9.895 9.948 252.990 2.536 2.763 26 7.091 6.536 6.814 27 4.700 4.768 4.734 28 0.7750.892 0.834 29 11.589 10.888 11.239 30 13.857 12.122 12.990

Table 8 shows levels of total cholesterol (TC), free cholesterol (FC)and cholesterol ester (CE) in the thoracic aorta at 12 weeks for the 30mice tested in the study. Mouse “sample” 1-15 are control mice (receivedvehicle only); mouse “sample” 16-20 are test mice (receivedaromatic-cationic peptide). As shown in Table 8, treatment witharomatic-cationic peptides lowers the total cholesterol, freecholesterol and cholesterol esters in the thoracic aorta.Aromatic-cationic peptides of the present disclosure are thereforeuseful in treating atherosclerosis and related signs, symptoms andcomplications of atherosclerosis.

TABLE 8 Thoracic Aorta Lipids Sample TC FC CE # μg/mg μg/mg μg/mgVehicle 1 37.3 8.3 28.9 Vehicle 2 28.7 7.2 21.5 Vehicle 3 23.5 9.2 14.2Vehicle 4 28.7 7.7 21.0 Vehicle 5 59.6 20.5 39.1 Vehicle 6 21.3 6.2 15.1Vehicle 7 37.6 6.8 30.8 Vehicle 8 19.0 5.0 14.0 Vehicle 9 27.1 7.0 20.1Vehicle 10 31.7 12.5 19.2 Vehicle 11 37.0 5.2 31.8 Vehicle 12 20.6 3.017.6 Vehicle 13 24.5 4.2 20.3 Vehicle 14 27.3 3.5 23.7 Vehicle 15 28.36.3 22.0 AVE 30.1 7.5 22.6 SEM 2.6 1.1 1.8 Peptide 16 21.4 7.6 13.8Peptide 17 6.7 4.0 2.7 Peptide 18 35.4 5.1 30.3 Peptide 19 17.7 3.0 14.7Peptide 20 38.8 8.1 30.7 Peptide 21 16.8 2.7 14.1 Peptide 22 8.1 3.5 4.6Peptide 23 20.1 14.6 5.6 Peptide 24 35.1 6.6 28.5 Peptide 25 9.4 3.8 5.6Peptide 26 24.9 3.6 21.3 Peptide 27 21.7 3.0 18.6 Peptide 28 7.9 3.3 4.6Peptide 29 19.0 7.6 11.4 Peptide 30 22.5 9.5 13.0 AVE 20.4 5.7 14.6 SEM2.6 0.9 2.5

Table 9 shows the total lesion area in the aortic root 300 μm across theaortic valve. Sample ID#1-15 are control mice (received vehicle only);Sample ID#16-20 are test mice (received aromatic-cationic peptide). Asshown in Table 9, treatment with aromatic-cationic peptides reducestotal lesion area. Aromatic-cationic peptides of the present disclosureare therefore useful in treating atherosclerosis and related signs,symptoms and complications of atherosclerosis.

TABLE 9 Total Lesion Area in Aortic Root 300 μm Across Aortic ValveSample Sample ID# Area(mm²) ID# Area(mm²) 1 325.02 16 323.88 2 250.92 17109.92 3 342.78 18 480.18 4 264.06 19 100.44 5 402.84 20 362.52 6 293.2821 259.38 7 408.48 22 185.04 8 323.88 23 310.38 9 429.54 24 401.46 10302.04 25 201.84 11 343.62 26 347.58 12 375.36 27 280.92 13 613.26 28117.96 14 376.14 29 427.8 15 225.36 30 331.5 AVE 351.77 282.72 SEM 24.1830.58

Table 10A-10D and FIGS. 4A-H show plasma lipid levels at t=0 weeks, 4weeks, 8 weeks, and 12 weeks. For each of the tables, total cholesterol(TC), free cholesterol (FC), cholesterol ester (CE), triglycerides(Trigs) and phospholipid (PL) is shown for each of the 15 control(sample #1-15) and 15 test animals (sample #16-30). Also provided areaverage values (AVE), and standard error of the mean (SEM). As show intables 10A-10D, and in FIGS. 4A-4H, at 4, 8 and 12 week time points,treatment with aromatic-cationic peptides reduces plasma totalcholesterol, VLDL-C, LDL-C, free cholesterol, cholesterol ester,triglycerides and phospholipid levels. Aromatic-cationic peptides of thepresent disclosure are therefore useful in treating atherosclerosis andrelated signs, symptoms and complications of atherosclerosis.

TABLE 10A Plasma Lipid Levels-Week 0 Spife (electrophoresis)ResultsSample mg/dL mg/dL mg/dL # TC HDL-C VLDL-C LDL-C FC CE Trigs PL 1 405 2225 358 136 269 84 255 2 546 63 39 444 159 387 174 357 3 304 28 19 258110 194 132 248 4 320 28 18 274 119 201 75 263 5 323 29 24 270 118 20596 277 6 302 34 25 242 120 182 165 277 7 381 38 24 319 140 241 167 328 8342 12 24 307 120 222 158 285 9 361 25 31 305 127 234 179 288 10 434 1521 398 139 295 151 316 11 431 5 19 406 142 289 253 295 12 564 19 33 513157 407 121 331 13 621 30 27 564 173 448 154 374 14 301 13 16 273 117184 103 264 15 536 12 27 498 162 374 109 299 AVE 411 25 25 362 136 275141 297 SEM 28 4 2 27 5 23 12 10 16 302 19 25 258 119 183 75 267 17 31834 23 260 121 197 88 267 18 376 32 37 307 134 242 117 308 19 302 37 20245 110 192 96 255 20 323 35 20 268 116 207 71 267 21 335 21 19 295 131204 138 306 22 369 38 27 304 129 240 88 298 23 550 41 36 474 184 366 167369 24 309 6 15 287 113 196 88 238 25 409 31 26 352 139 270 136 320 26431 26 25 380 134 297 151 313 27 552 39 31 481 170 382 191 332 28 457 2224 410 151 306 188 343 29 490 29 28 432 159 331 154 338 30 808 8 29 773220 588 353 413 AVE 422 28 26 368 142 280 140 309 SEM 35 3 2 35 8 28 1812

TABLE 10B Plasma Lipid Levels-Week 4 Spife (electrophoresis) ResultsSample mg/dL mg/dL mg/dL # TC HDL-C VLDL-C LDL-C FC CE Trigs PL 1 119539 54 1101 456 739 82 634 2 1103 31 51 1022 394 709 54 558 3 1114 11 511052 424 690 44 540 4 876 18 37 822 328 548 59 436 5 1124 11 58 1058 444680 18 480 6 913 23 27 863 332 581 62 456 7 1052 25 48 978 382 670 54528 8 908 15 31 862 352 556 72 460 9 948 16 38 894 372 576 72 470 10 85020 37 794 338 512 64 420 11 1245 32 34 1181 502 743 75 590 12 1274 42 321200 582 692 128 690 13 1267 33 22 1212 524 743 98 658 14 1116 32 301054 406 710 57 540 15 1177 32 29 1115 510 667 57 568 AVE 1078 25 391014 423 654 66 535 SEM 38 3 3 36 20 20 6 21 16 799 18 35 745 308 491 18422 17 924 20 37 866 344 580 39 482 18 976 20 37 918 354 622 28 452 191139 0 19 1120 408 731 85 560 20 1038 13 44 982 412 626 59 526 21 673 718 648 256 417 18 320 22 940 23 23 893 366 574 46 506 23 1046 32 39 975386 660 108 556 24 835 20 44 770 320 515 59 446 25 822 15 41 766 286 53633 416 26 1026 32 28 966 396 630 44 516 27 1223 45 31 1147 462 761 67580 28 935 32 30 874 354 581 44 510 29 1248 44 32 1171 516 732 80 666 301337 49 35 1254 636 701 144 750 AVE 997 25 33 940 387 610 58 514 SEM 474 2 45 25 25 9 27

TABLE 10C Plasma Lipid Levels-Week 8 Spife (electrophoresis) ResultsSample mg/dL mg/dL mg/dL # TC HDL-C VLDL-C LDL-C FC CE Trigs PL 1 159026 76 1488 418 1172 79 548 2 1342 10 107 1225 352 990 58 518 3 1270 4 661201 346 924 39 482 4 1611 40 82 1489 394 1217 60 526 5 1610 16 107 1488420 1190 79 536 6 902 19 51 831 278 624 53 410 7 1484 28 54 1402 4081076 89 564 8 986 23 75 887 288 698 58 380 9 1491 24 70 1397 394 1097 65554 10 1568 23 86 1459 388 1180 63 534 11 1667 30 149 1488 470 1197 113594 12 1842 86 143 1613 574 1268 209 738 13 1695 62 137 1496 448 1247142 534 14 1719 22 106 1591 454 1265 96 570 15 765 18 39 708 250 515 41396 AVE 1436 29 90 1318 392 1044 83 526 SEM 83 5 9 74 21 64 11 23 161262 42 101 1119 322 940 34 416 17 1004 34 73 897 294 710 31 456 18 150527 86 1392 386 1119 34 474 19 1183 33 79 1071 318 865 51 448 20 1545 19112 1415 392 1153 48 516 21 943 8 34 901 290 653 31 424 22 1008 45 88875 294 714 51 480 23 1473 16 79 1378 388 1085 53 488 24 1023 32 69 922308 715 43 436 25 956 41 59 856 286 670 75 440 26 1435 25 93 1317 3621073 63 556 27 1414 19 74 1320 366 1048 39 516 28 1147 41 80 1025 308839 34 470 29 1534 43 109 1382 404 1130 67 540 30 1797 57 119 1621 5301267 195 728 AVE 1282 32 84 1166 350 932 57 493 SEM 69 3 6 64 17 53 1020

TABLE 10D Plasma Lipid Levles-Week 12 Spife (electrophoresis) ResultsSample mg/dL mg/dL mg/dL # TC HD L-C VLDL-C LDL-C FC CE Trigs PL 1 111539 36 1041 351 764 42 444 2 637 15 35 587 225 412 25 336 3 803 23 24 756269 533 25 381 4 1243 48 105 1090 380 863 31 477 5 1335 9 69 1257 429907 11 499 6 874 28 36 811 307 567 31 427 7 1077 35 43 998 346 731 20445 8 827 23 44 759 277 550 20 379 9 1205 27 43 1135 376 829 24 586 10910 36 44 830 306 604 16 417 11 1220 50 62 1108 371 848 93 497 12 175456 59 1640 543 1211 60 700 13 1710 17 83 1609 558 1152 97 689 14 1085 3876 972 359 725 51 422 15 750 12 31 707 269 482 60 396 AVE 1103 30 531020 358 745 40 473 SEM 84 4 6 79 24 60 7 28 16 898 38 39 821 291 607 27366 17 540 20 35 485 205 335 49 318 18 884 10 21 854 287 597 10 320 19937 41 43 852 296 640 37 432 20 1420 21 71 1328 418 1002 18 491 21 719 521 693 242 477 39 321 22 674 31 32 611 229 445 39 363 23 745 20 33 692269 476 37 394 24 990 2 26 962 315 675 13 374 25 999 40 63 896 312 68725 423 26 1379 28 76 1275 419 960 74 536 27 1255 1 36 1218 386 869 89521 28 558 23 30 505 219 339 97 340 29 1317 31 83 1203 413 904 112 57530 1736 2 46 1688 535 1201 158 741 AVE 1003 21 44 939 322 681 55 434 SEM90 4 5 88 24 66 11 31

Results are further shown in FIGS. 1-3. FIG. 1A shows that at 12 weeks,the % lesion area is lower in the treated (dark bar) versus untreated(white bars) group. Thus there is a decrease in plaque present in thethoracic aorta of treated versus untreated subjects. FIG. 1B is aphotograph of lesions on the vehicle only aorta versus the treatedaorta. As shown in the bar graph in FIG. 2, the level of thoracic aortaplaque cholesterol content (TC=total cholesterol; FC=free cholesteroland CE=cholesterol ester) is lower in the treated (dark bars) versusuntreated (vehicle, white bars) group at 12 weeks. FIG. 3 shows that, at12 weeks, mean lesion area is lower in the treated group versus theuntreated (vehicle) group. Accordingly, the aromatic-cationic peptidesof the present disclosure are useful for decreasing the amount ofatherosclerotic plaque in both the aorta and aortic root, and fordecreasing the plaque cholesterol content. Thus, the aromatic-cationicpeptides of the present disclosure are useful for treating or preveingatherosclerosis and related signs, symptoms and complications ofatherosclerosis.

Example 2 Effects of Aromatic-Cationic Peptides in Conjunction with anAntihyperlipidemic Agent (e.g., Statins) to Protect AgainstAtherosclerosis and Lower Cholesterol Levels in a Mouse Model

The effects of aromatic-cationic peptides in protecting againstatherosclerosis, in conjunction with one or more antihyperlipidemicagents, in this example, statins, in a mouse model are investigated asfollows.

Mice are treated as described in Example 1. That is, Apoprotein Edeficient mice as described in Example 1 are used in the study. Aninitial total cholesterol measurement is made on the mice, and the miceare grouped into groups of 15 to match the total cholesterolmeasurements. The groups are fed a “western diet” (40 kcal % butterfat,0.15% [wt/wt] cholesterol). Starting at t=0, the control group of 15mice receives vehicle only, while the test groups of 15 mice receivearomatic-cationic peptide and one or more statins. Body weights of themice are recorded weekly, and mortality checks are performed daily.

The aromatic-cationic peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ (sterilelyophilized powder) is tested. Each group of test mice receives asingle, daily dose of the peptide, subcutaneously at 1, 3, 5 or 10mg/kg, and also receives, simultaneously atorvastatin, fluvastatin,lovastatin, pravastatin or rosuvastatin at 0.1, 0.5, 0.75 or 1 mg/kg.Control mice receive vehicle only. The injections continue for 12 weeks,at which time the mice are sacrificed and analyzed as described inExample 1.

Plasma lipid analysis using an autoanalyzer includes an evaluation oftotal cholesterol (TC), triglycerides (Trigs), phospholipids (PL), freecholesterol (FC), and cholesterol ester (CE, by calculation). Gelelectrophoresis is used to measure levels of high-density lipoproteincholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), andvery low-density lipoprotein cholesterol (VLDL-C).Histopathology/histomorphometery is performed as described above inExample 1.

Results: It is anticipated that mice receiving both the peptide and thestatin will show decreased levels of total cholesterol, freecholesterol, triglyceride, phospholipid, cholesterol ester, LDL-C andVLDL-C as well as a decrease in the lesions as compared to subjectsreceiving vehicle only. It is also anticipated that in some instances,subjects receiving the combination treatment (aromatic-cationic peptideplus statin) will exhibit a synergy between the two drugs, such that alower dose of peptide, the statin or both will achieve desired results,e.g., lowered levels of total cholesterol, free cholesterol,triglyceride, phospholipid, cholesterol ester, LDL-C and VLDL-C and/orlesions.

Accordingly, it is anticipated that the results will further demonstratethat the aromatic-cationic peptides of the present disclosure, alone orin combination with one or more statins, will be useful for treatingatherosclerosis, and signs, symptoms or complications ofatherosclerosis, including but not limited to increased totalcholesterol, free cholesterol, triglyceride, phospholipid, cholesterolester, LDL-C and VLDL-C and increased atherosclerotic lesions.

Example 3 Aromatic-Cationic Peptides Increase Coenzyme Q10 Levels

Fibroblasts were treated with an aromatic-cationic peptide of thepresent disclosure, and levels of coenzyme Q10 were evaluated.

Human skin fibroblasts from normal subjects were incubated understandard tissue culture medium containing high glucose levels to supportanaerobic conditions. At approximately 90% confluence, cells wereincubated in the presence of 10 nM D-Arg-2′6′-Dmt-Lys-Phe-NH₂ for aperiod of either one day (24 h) or five days as shown in Table 11 below.The culture medium was not changed during the incubation period. Cellswere harvested and the cellular level of CoQ was determined usingmethods known in the art. Data represents the average n=3-6.

TABLE 11 Treatment of fibroblasts with aromatic-cationic peptide Time ofPeptide Cells Medium Peptide Amount Treatment fibroblast DMEM 0 0 cellsfibroblast DMEM 10 nM 16-24 hours cells fibroblast DMEM 10 nM 5 dayscells

Results are shown in FIG. 5. As shown in FIG. 5, exposure to thearomatic-cationic peptides of the present disclosure increased coenzymeQ10 levels in fibroblast cells. Accordingly, the aromatic-cationicpeptides of the present disclosure are useful for increasing coenzymeQ10 levels in subjects in need thereof. For example, thearomatic-cationic peptides of the present disclosure are useful forincreasing coenzyme Q10 levels in subjects taking one or more statindrugs and/or in subjects suffering from a disease or conditionscharacterized by, or caused by low (e.g., below normal or controllevels) coenzyme Q10 levels. The aromatic-cationic peptides of thepresent disclosure are useful to treat, prevent or ameliorate the signsand/or symptoms of diseases or conditions characterized by low (e.g.,below normal or control levels) coenzyme Q10 levels.

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method for treating atherosclerosis in amammalian subject in need thereof, the method comprising administeringan effective amount of (i) a peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or apharmaceutically acceptable salt thereof and (ii) an antihyperlipidemicdrug, wherein the peptide and the antihyperlipidemic drug are chemicallylinked.
 2. The method of claim 1, wherein the antihyperlipidemic drug isselected from the group consisting of: atorvastatin, simvastatin,pravastatin, fluvastatin, lovastatin, pitavastatin, rosuvastatin,clinofibrate, clofibrate, simfibrate, fenofibrate, bezafibrate,colestimide, colestyramine, ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin).
 3. The method of claim 1, wherein theantihyperlipidemic drug is a statin.
 4. The method of claim 3, whereinthe statin is selected from the group consisting of: ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin).
 5. The method of claim 1, wherein treatmentincludes decreasing the size or number of atherosclerotic plaques in thesubject, and/or decreasing the cholesterol content of an atheroscleroticplaque in the subject.
 6. A method for preventing atherosclerosis in amammalian subject in need thereof, the method comprising administeringan effective amount of (i) a peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or apharmaceutically acceptable salt thereof and (ii) an antihyperlipidemicdrug, wherein the peptide and the antihyperlipidemic drug are chemicallylinked.
 7. The method of claim 6, wherein the antihyperlipidemic drug isselected from the group consisting of: atorvastatin, simvastatin,pravastatin, fluvastatin, lovastatin, pitavastatin, rosuvastatin,clinofibrate, clofibrate, simfibrate, fenofibrate, bezafibrate,colestimide, colestyramine, ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin).
 8. The method of claim 6, wherein theantihyperlipidemic drug is a statin.
 9. The method of claim 8, whereinthe statin is selected from the group consisting of: ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin).
 10. The method of any one of claims 6-9,wherein the subject is predisposed to atherosclerosis.
 11. A method forameliorating the signs, symptoms or complications of atherosclerosis,the method comprising administering an effective amount of (i) a peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable salt thereofand (ii) an antihyperlipidemic drug, wherein the peptide and theantihyperlipidemic drug are chemically linked.
 12. The method of claim11, wherein the signs, symptoms or complications of atherosclerosisinclude one or more selected from the group consisting of: elevatedlevels total cholesterol, VLDL cholesterol, LDL cholesterol, freecholesterol, cholesterol ester, phospholipids, triglycerides, andatherosclerotic lesions.
 13. The method of claim 11, wherein theantihyperlipidemic drug is selected from the group consisting of:atorvastatin, simvastatin, pravastatin, fluvastatin, lovastatin,pitavastatin, rosuvastatin, clinofibrate, clofibrate, simfibrate,fenofibrate, bezafibrate, colestimide, colestyramine, ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin).
 14. The method of claim 11, wherein theantihyperlipidemic drug is a statin.
 15. The method of claim 14, whereinthe statin is selected from the group consisting of: ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin).
 16. The method of claim 11, whereinameliorating the signs, symptoms or complications of atherosclerosisincludes decreasing the size or number of atherosclerotic plaques in thesubject, and/or decreasing the cholesterol content of an atheroscleroticplaque in the subject.
 17. A method for delaying onset, ameliorating oreliminating statin side effects in a subject in need thereof, the methodcomprising administering an effective amount of a peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof, wherein the peptide is chemically linked to the statin.
 18. Themethod of claim 17, wherein the statin side effect includes one or moreof myopathy, rhabdomyolysis, kidney failure, diabetes, memory loss,decreased coenzyme Q10 levels and mitochondrial dysfunction.
 19. Themethod of claim 17, wherein the statin is selected from the groupconsisting of: atorvastatin, simvastatin, pravastatin, fluvastatin,lovastatin, pitavastatin, rosuvastatin, ADVICOR® (niacinextended-release/lovastatin), ALTOPREV® (lovastatin extended-release),CADUET® (amlodipine and atorvastatin), CRESTOR® (rosuvastatin),JUVISYNC® (sitagliptin/simvastatin), LESCOL® (fluvastatin), LESCOL XL(fluvastatin extended-release), LIPITOR® (atorvastatin), LIVALO®(pitavastatin), MEVACOR® (lovastatin), PRAVACHOL® (pravastatin), SIMCOR®(niacin extended-release/simvastatin), VYTORIN® (ezetimibe/simvastatin),and ZOCOR® (simvastatin).
 20. A method for increasing statin dosage in asubject in need thereof comprising: administering an effective amount ofa statin at a first dosage level, and an aromatic-cationic peptide,wherein the statin and the aromatic-cationic peptide are chemicallylinked; evaluating the subject for side-effects characteristic of thestatin, wherein the side-effects in the subject are reduced or absent ascompared to a control subject administered the statin and not thearomatic cationic peptide; administering a statin at a second dosagelevel, wherein the second dosage level is higher than the first statindosage level.
 21. The method of claim 20, wherein the peptide isD-Arg-2′6′Dmt-Lys-Phe-NH₂.
 22. The method of claim 20, wherein thestatin comprises LIPITOR® or CRESTOR®.
 23. The method of claim 20,wherein the side effect characteristic of the statin includes one ormore of myopathy, rhabdomyolysis, kidney failure, diabetes, memory loss,decreased coenzyme Q10 levels and mitochondrial dysfunction.
 24. Themethod of any one of claims 1-23, wherein the pharmaceuticallyacceptable salt comprises acetate salt or trifluoroacetate salt.
 25. Themethod of any one of claims 1-25, wherein the peptide and theantihyperlipidemic drug or the statin are linked using a labile linkagethat is hydrolyzed in vivo to release the peptide and theantihyperlipidemic drug or the statin.
 26. The method of claim 26,wherein the labile linkage comprises an ester linkage, a carbonatelinkage, or a carbamate linkage.
 27. The method of any one of claim 1, 6or 11, wherein the antihyperlipidemic drug is an anti-PCSK9 antibody.28. The method of claim 27, wherein the peptide and the anti-PCSK9antibody are linked using a bifunctional protein coupling agent.
 29. Themethod of claim 28, wherein the bifunctional protein coupling agent isN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), a bifunctional derivative of an imidoester, anactive ester, an aldehyde, a bis-azido compound, a bis-diazoniumderivative, a diisocyanate, or a bis-active fluorine compound.
 30. Themethod of claim 27, wherein the peptide and the anti-PCSK9 antibody arelinked using a labile linkage.
 31. The method of claim 30, wherein thelabile linkage comprises an ester linkage, a carbonate linkage, or acarbamate linkage.
 32. The method of any one of claim 1, 6 or 11,wherein the antihyperlipidemic agent is an Apo-B antisenseoligonucleotide.
 33. The method of claim 32, wherein the peptide and theApo-B antisense oligonucleotide are linked using a labile linkage. 34.The method of claim 33, wherein the labile linkage comprises an esterlinkage, a carbonate linkage, or a carbamate linkage.